High affinity IP3-binding polypeptide

ABSTRACT

The present invention provides a high affinity polypeptide having a binding activity to inositol 1,4,5-trisphosphate, to a gene encoding the polypeptide, to a recombinant vector including the gene, to a transformant including the vector and to a method for producing the high affinity polypeptide having a binding activity to inositol 1,4,5-trisphosphate.

FIELD OF THE INVENTION

[0001] The present invention relates to a high affinity polypeptide having a binding activity to inositol 1,4,5-trisphosphate, to a gene encoding the polypeptide, to a recombinant vector including the gene, to a transformant including the vector and to a method for producing the high affinity polypeptide having a binding activity to inositol 1,4,5-trisphosphate.

BACKGROUND OF THE INVENTION

[0002] Inositol 1,4,5-trisphosphate (hereinafter, also referred to as “IP₃”) is one of second messengers which are produced by inositol phospholipid metabolism activated in response to an extracellular stimului such as hormones, growth factors, neurotransmitters or the like. IP₃ is a substance that induces the increase of intracellular calcium concentration. The IP₃-induced calcium increase is a crucial and highly universal signal transmission mechanism that is involved in many cell functions in a wide variety of animals. For example, IP₃ controls many physiological functions such as fertilization, blastogenesis, development and differentiation, cell growth, secretion, immune system, muscle contraction, and cranial nerve functions (gustation, vision, memory, learning, etc.) in diverse organisms, for example, invertebrata such as nematoda (nemathelminthes), Drosophila (arthropoda) and cuttlefish (mollusca), and vertebrata such as mouse and human.

[0003] On the molecular level, this mechanism is initiated by the binding between an IP₃ and its target, an IP₃ receptor. Specifically, when the IP₃ binds to the IP₃ receptor (a calcium channel susceptible to IP₃) present in an intracellular calcium-storing site (endoplasmic reticulum, etc.), the channel opens and releases calcium from the calcium-storing site into the cytoplasm, thereby controlling the activities of calcium-dependent proteins and enzymes.

[0004] Heparin, adenophostin (a kind of fungal metabolite) and Xestospongin (a kind of sponge metablite) are examples of substances that might affect the signal transmission by the IP₃-induced calcium. However, although heparin inhibits the binding between the IP₃ and the IP₃ receptor, its specificity is low since there are various targets in the cell. Adenophostin is an antagonistic agonist of the binding between the IP₃ and the IP₃ receptor, and is a powerful activator of the IP₃ receptor channel. However, its use is limited since its yield from fungus is low and it cannot transport across the membrane. Xestospongin has recently been reported as an inhibitor of the IP₃ receptor channel that does not influence the binding of IP₃. Again, its yield is low and there are still questions remaining as to its specificity. Thus, currently, there is almost no substance that is considered to effectively act on IP₃-induced calcium signal transmission. In particular, there has been no substance or system that inhibits IP₃-induced calcium signal transmission by specifically trapping IP₃ that has increased on the cell level.

SUMMARY OF THE INVENTION

[0005] The present invention provides a high affinity polypeptide having a binding activity to inositol 1,4,5-trisphosphate, a gene encoding the polypeptide, a recombinant vector containing the gene, a transformant containing the vector and a method for producing the high affinity polypeptide having a binding activity to inositol 1,4,5-trisphosphate.

[0006] In order to solve the above-described problem, the present inventors have gone through intensive studies and have succeeded in isolating a high affinity polypeptide having an extremely high binding activity to IP₃ from a protein including a part of the N-terminal amino acid region of an IP₃ receptor.

[0007] The present invention provides a recombinant polypeptide of the following (a), (b) or (c):

[0008] (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 2;

[0009] (b) a polypeptide comprising an amino acid sequence having deletion, substitution or addition of at least one amino acid in the amino acid sequence shown in SEQ ID NO: 2, and having a high affinity binding activity to inositol 1,4,5-trisphosphate; or

[0010] (c) a polypeptide having at least 70% homology with the amino acid sequence shown in SEQ ID NO: 2, and having a high affinity binding activity with inositol 1,4,5-trisphosphate.

[0011] The present invention also provides a gene coding for a polypeptide of the above (a), (b) or (c); or a gene coding for a polypeptide having at least 70% homology with the gene and having a high affinity binding activity with inositol 1,4,5-trisphosphate.

[0012] The present invention further provides a gene comprising DNA of the following (d) or (e):

[0013] (d) DNA of a nucleotide sequence shown in SEQ ID NO: 1; or

[0014] (e) DNA of a nucleotide sequence having at least 70% homology with the DNA of the nucleotide sequence shown in SEQ ID NO: 1, and coding for a polypeptide having a high affinity binding activity with inositol 1,4,5-trisphosphate.

[0015] The present invention provides a recombinant vector comprising any one of the above-described genes.

[0016] The present invention also provides a transformant comprising the above recombinant vector.

[0017] The present invention further provides a method for producing any one of the above-mentioned polypeptides, the method comprising: culturing the above-mentioned transformant; and collecting, from the obtained culture, a polypeptide having a high affinity binding activity to inositol 1,4,5-trisphosphate.

[0018] This and other advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description with reference to the accompanying figures.

[0019] This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 10-242207 which is a priority document of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 shows the structures of IP₃ sponges;

[0021] FIGS. 2A-2C show high expression and IP₃-binding activity of T604;

[0022] FIGS. 3A-3C are graphs showing the IP₃-binding activities of the IP₃ sponges;

[0023]FIG. 4 is a graph showing a curve of IP₃-binding inhibition depending on the IP₃ sponge concentration;

[0024] FIGS. 5A-5F are graphs showing the effects of low-affinity G224-m30 and GST on IP₃-induced Ca²⁺ release;

[0025] FIGS. 6A-6G are graphs showing the effect of high affinity IP₃ sponge G224 on inhibition of IP₃-induced Ca²⁺ release; and

[0026]FIG. 7 is a plot diagram showing an IP₃-induced Ca²⁺ release depending on the concentration of the high affinity IP₃ sponge G224.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Hereinafter, the present invention will be described in more detail.

[0028] A polypeptide of the present invention specifically binds to IP₃with a very high affinity, and includes a part (a cut) of N-terminal amino acid region of the natural IP₃ receptor (thus also referred to as a cut-type polypeptide). The polypeptide of the invention is often referred to as a high affinity IP₃-binding polypeptide.

[0029] 1. Cloning a Gene Coding for the IP₃ Receptor

[0030] In order to obtain a high affinity IP₃-binding polypeptide of the invention, a gene encoding the natural IP₃ receptor protein is cloned. The nucleotide sequence of the IP₃ receptor gene is already known (Nucleic Acid Res. 17: 5385-5386, 1989; Nature 342:32-38, 1989). The gene may, for example, be prepared according to the following gene engineering procedure.

[0031] (i) Preparation and screening of cDNA library encoding the IP₃ receptor

[0032] A known procedure may be employed to prepare mRNA of the IP₃ receptor. For example, total RNA is obtained by treating a tissue or a cell from a mouse brain with a guanidine reagent, a phenol reagent or the like. Then, poly(A)+RNA(mRNA) is obtained according to an affinity column method or a batch method using poly (U) sepharose, etc. By using the obtained mRNA as a template as well as oligo dT primer and reverse transcriptase, a single-stranded cDNA is synthesized. Based on the single-stranded cDNA, a double-stranded cDNA is synthesized and introduced into a suitable cloning vector to prepare a recombinant vector to transform E.coli or the like. The transformant is selected based on indices such as tetracycline and ampicillin resistance, thereby obtaining a cDNA library.

[0033] The transformation of E.coli may be conducted according to the method of Hanahan [Hanahan, D., J. Mol. Biol. 166:557-580 (1983)]. Specifically, the recombinant vector is added to a prepared competent cell under the presence of calcium chloride, magnesium chloride or rubidium chloride. When a plasmid is used as the vector, it should contain a gene resistant to drugs such as tetracycline and ampicillin. Besides plasmids, a cloning vector such as λ phage may also be used.

[0034] The thus-obtained transformant is screened for strains with the DNA of interest by, for example, “expression cloning” through immunoscreening using an antibody, or by polymerase chain reaction (PCR) using a primer synthesized from a known sequence.

[0035] The thus-obtained DNA fragment or DNA amplified fragment coding for the antibody epitope is labeled with ³²P, ³⁵S, biotin or the like to be used as a probe for hybridizing with the transformant DNA denatured and bound on a nitrocellrose filter. Then, the obtained positive strains may be screened for the target DNA fragment.

[0036] (ii) Determination of the nucleotide sequence

[0037] The obtained clone is determined for its nucleotide sequence. The nucleotide sequence may be determined according to a known method such as Maxam-Gilbert chemical modification method, dideoxynucleotide chain termination method using M13 phage. Generally, the sequence is determined using an automatic DNA sequencer (e.g., Perkin-Elmer 373A DNA sequencer).

[0038] The nucleotide sequence of the natural (full-length) gene coding for the IP₃ receptor and the full-length amino acid sequence of the IP₃ receptor are shown in SEQ ID NOS. 3 and 4, respectively.

[0039] 2. Design and Synthesis of a Gene Coding for a High Affinity IP₃-Binding Polypeptide of the Invention

[0040] (i) Design and synthesis of a gene coding for a high affinity IP₃-binding polypeptide

[0041] A high affinity IP₃-binding polypeptide of the invention includes a cut of N-terminal amino acid region, that is, Amino acids 579 to at least 800, preferably Amino acids 579 to at least 734, of the amino acid sequence of the full-length IP₃ receptor protein (SEQ ID NO:4). According to the present invention, this cut-type polypeptide is also referred to as an IP₃ sponge (FIG. 1). Due to this cut, the polypeptide (IP₃ sponge) of the invention gains a very strong specific binding ability to IP₃ (high affinity IP₃-binding activity).

[0042] Herein, the phrase “high affinity” is used in the situation where the IP₃ sponge has an IP₃ affinity that is about 100 to 1,000 times (preferably 500 to 1,000 times) higher than that of the natural IP₃ receptor.

[0043] According to the present invention, the IP₃ sponge also includes at least the amino acid sequence shown in SEQ ID NO: 2, which corresponds to Amino acids 226-578 of the amino acid sequence of SEQ ID NO: 4. Herein, this region is referred to as a “core” region.

[0044] Based on the above-described facts, the length of the fragment of the invention and the length of the DNA coding for the fragment can be determined at ones discretion providing that the high affinity IP₃-binding activity is maintained. The fragment may include, for example, Amino acids 224-604 of the amino acid sequence of SEQ ID NO: 4 (encoded by Nucleotides 670-1812 of the nucleotide sequence of SEQ ID NO: 3); Amino acids 1-604 of the amino acid sequence of SEQ ID NO: 4 (encoded by Nucleotides 1-1812 of the nucleotide sequence of SEQ ID NO: 3); or Amino acids 1-734 of the amino acid sequence of SEQ ID NO: 4 (encoded by Nucleotides 1-2200 of the nucleotide sequence of SEQ ID NO: 3).

[0045] These fragments are obtained through PCR using primers that are designed based on nucleotide regions of the nucleotides shown in SEQ ID NO. 3 outside the regions of the respective fragments, as well as the DNA coding for the natural IP₃ receptor (SEQ ID NO: 3, Nucleic Acid Res. 17: 5385-5386, 1989; Nature 342: 32-38, 1989) as a template.

[0046] (ii) Preparation of a gene encoding a mutant-type IP₃ sponge of the invention (mutant-type IP₃ gene)

[0047] According to the present invention, the amino acid sequence of the IP₃ sponge may, at least partially, be introduced with a mutation. Such a mutant-type IP₃ sponge is also contemplated as the IP₃ sponge of the present invention. A mutation is introduced into the amino acid sequence, by mutating the nucleotide sequence of the gene coding for the amino acid sequence of the IP₃ sponge.

[0048] The mutation is introduced into the gene according to a known method such as Kunkel method, Gapped duplex method or any method equivalent thereof. For example, site-directed mutatagenesis may be employed in which a mutant oligonucleotide is used as a primer (Yoshikawa, F. et al., J. Biol. Chem. 271: 18277-18284, 1996). Alternatively, a mutation may be introduced by using a mutagenesis kit such as Mutant-K (Takara), Mutant-G (Takara) and a series of LA PCR in vitro Mutagenesis kits (Takara).

[0049] First, based on the nucleotides of the gene coding for the IP₃ sponge of the invention (also referred to as an “IP₃ sponge gene”), a primer is synthesized such that the primer includes a mutated nucleotide or site and about 10 nucleotides flanking the mutated nucleotide or site. Using this primer as well as the IP₃ sponge gene as a template, PCR reaction is conducted. The resultant is purified and then treated with a suitable restriction enzyme, thereby obtaining the mutant-type IP₃ sponge gene of interest.

[0050] (iii) Determination of the nucleotide sequences

[0051] The nucleotide sequence of the genes obtained through (i) and (ii) is determined. The determination is conducted by a known method such as Maxam-Gilbert chemical modification method, dideoxynucleotide chain termination method using M13 phage, or any other method. Generally, an automatic sequencer (e.g., 373A DNA sequencer produced by Perkin-Elmer) is used.

[0052] A nucleotide sequence of an IP₃ sponge gene of the invention and an amino acid sequence of the IP₃ sponge of the invention are shown in SEQ ID NOS: 1 and 2, respectively. The polypeptide of this amino acid sequence may include at least one deletion, substitution, addition or the like as long as it has a high affinity with IP₃ and has an activity of specifically binding to IP₃.

[0053] For example, at least one, preferably about 1 to 10, more preferably 1 to 5 of the amino acids in the core region (the amino acid sequence shown in SEQ ID NO: 2) may be deleted; at least one, preferably about 1 to 10, more preferably 1 to 5 amino acids may be added to the amino acid sequence of the core region; or at least one, preferably 1 to 10, more preferably 1 to 5 of the amino acids in the core region may be replaced with other amino acids.

[0054] The polypeptide of the present invention is not limited by the length of the amino acid sequence as long as the amino acid sequence contains the amino acid sequence of the core region, and a cut of N-terminal Amino acids 579 to at lease 800, preferably N-terminal Amino acids 579 to at least 734 of the natural-type IP₃ receptor (SEQ ID NO: 4). For example, Amino acids 224-604 (polypeptide “G224”) of the amino acid sequence shown in SEQ ID NO: 4, and the gene encoding G224 are also contemplated as the IP₃ sponge and the IP₃ sponge gene of the invention, respectively.

[0055] The polypeptide G224 may have a mutation of at least one, preferably about 1 to 10, more preferably 1 to 5 amino acids. Thus, the IP₃ sponge of the invention may include an amino acid sequence where Lysine at Position 508 of the amino acid sequence G244 is replaced with alanine (mutation “m30”) or where arginine at Position 441 of the amino acid sequence G244 is replaced with glutamin (mutation “m49”) (FIG. 1). Herein, the numbers indicating the positions of the amino acids are based on the amino acid sequence shown in SEQ ID NO: 4 (e.g., Position 1 is the first amino acid of SEQ ID NO: 4).

[0056] A polypeptide including an amino acid sequence having 70% or more homology with the core region (SEQ ID NO: 2), and having a high affinity binding activity with inositol 1,4,5-trisphosphate is also contemplated as the present invention.

[0057] Also contemplated as the present invention is a gene coding for the polypeptide having the above-described mutation in its amino acid sequence, and having a high affinity binding activity with IP₃ receptor. In addition, a nucleotide sequence coding for the amino acids included in the IP₃ sponge of the present invention, and a degenerate isomer coding for the same polypeptide with different degenerate codons are also contemplated as the genes of the invention. Also contemplated as the present invention is DNA having at least 70% homology with the nucleotide sequence of these genes, for example, DNA of other type belonging to the IP₃ receptor gene family that codes for a region corresponding to the polypeptide of the present invention.

[0058] Once the nucleotide sequence of the gene of the present invention is determined, the gene may be obtained by PCR using a primer that is synthesized chemically or that is synthesized from the determined nucleotide sequence.

[0059] 3. Preparation of Recombinant Vector and Transformant Containing IP₃ Sponge Gene of the Invention

[0060] (i) Preparation of recombinant vector

[0061] A recombinant vector of the invention may be obtained by ligating (inserting) the IP₃ sponge gene of the invention to (into) a suitable vector. The vector for inserting the gene of the invention is not limited to a specific one as long as it is replicable in a host cell. Examples of such vector include but not limited to plasmid DNA and phage DNA.

[0062] The plasmid DNA is, for example, plasmid from E.coli (e.g., pET-3a, pBR322, pBR325, pUC118, pUC119, etc.), plasmid from bacillus (e.g., pUB110, pTP5, etc.), or plasmid from yeast (e.g., YEp13, YEp24, YCp50, etc.). The phage DNA is, for example, λ phage. Similarly, an animal virus vector such as retrovirus, adenovirus or vaccinia virus vectors, or an insect virus vector such as a baculovirus vector may also be used. A fusion plasmid in which GST, GFP, His-tag, Myc-tag or the like is linked with each other may also be used (e.g., pGEX-2T, pEGFP-N3).

[0063] To insert the gene of the invention into the vector, first, the purified DNA is cleaved with suitable restriction enzymes. Then, the cleaved fragment is inserted into a restriction site or a multicloning site of the suitable vector DNA.

[0064] The gene of the present invention should be integrated into the vector such that the gene can function. If desired, the vector of the invention may include, other than the gene of the invention and the promoter, for example, a cis-element (e.g., an enhancer), a splicing signal, a poly(A) tail signal, a selective marker, and a ribosome binding sequence (SD sequence). Examples of the selective marker include a dihydrofolate reductase gene, an ampicillin-resistant gene and a neomycin-resistant gene.

[0065] (ii) Preparation of transformant

[0066] A transformant of the invention may be obtained by introducing the recombinant vector of the invention into a host cell in such a manner that the gene of interest is capable to be expressed. The host cell is not limited to a specific one as long as it can express the gene of the present invention. Bacteria such as genus Escherichia (e.g., Escherichia coli), genus Bacillus (e.g., Bacillus subtilis), genus Pseudomonas (e.g,. Pseudomonas putida), yeast such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, animal cells (e.g., COS, CHO, HEK293, PC12 cells), and insect cells (e.g., Sf9 and Sf21) are exemplified.

[0067] When a bacterium such as E.coli is used as the host, it is preferable that the recombinant vector of the present invention is capable of autonomous replication in the host and that it includes a promoter, a ribosome binding sequence, the gene of the invention and a transcription termination sequence. The recombinant vector may also include a gene for controlling the promoter.

[0068] As the E.coli, E.coli BL21, JM109 and HB101 are exemplified and as bacillus, Bacillus subtilis MI 114 and 207-21 are exemplified.

[0069] Any promoter may be used as long as it can be expressed in a host cell like E.coli. For example, a promoter derived from E.coli or phage, e.g., trp promoter, lac promoter, p_(L) promoter or p_(R) promoter, may be used. Artificially designed and modified promoter like tac promoter may also be used.

[0070] The recombinant vector may be introduced into the host bacterium according to any method for introducing DNA into a bacterium. For example, calcium ion method (Cohen, S. N. et al., Proc. Natl. Acad. Sci., USA, 69: 2110-2114 (1972)) and an electroporation method may be employed.

[0071] An yeast such as Saccharomyces cerevisiae, Schizosaccharomyces pombe or Pichia pastoris may also be used as the host. In this case, the promoter may be any promoter that can be expressed in the yeast. Examples of such promoter include but not limited to gall promoter, ga110 promoter, heat shock protein promoter, MF 1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and AOX1 promoter.

[0072] The recombinant vector may be introduced into the yeast by any method for introducing DNA into an yeast. For example, electroporation method (Becker, D. M. et al., Methods Enzymol., 194, 182-187 (1990)), spheroplast method (Hinnen, A. et al., Proc. Natl. Acad. Sci., USA, 75, 1929-1933 (1978)), or lithium acetate method (Itoh, H., J. Bacteriol., 153, 163-168 (1983)) may be employed.

[0073] An animal cell such as simian cell (e.g., COS-7, Vero), Chinese hamster ovary cell (CHO cell), mouse L cell, rat cell (e.g., GH3, PC12 or NG108-15) or human cell (e.g., FL, HEK293, HeLa or Jurkat) may also be used as the host. As a promoter, for example, SR promoter, SV40 promoter, LTR promoter or β-actin promoter may be used. Other than these promoters, an early gene promoter of human cytomegalovirus may also be used.

[0074] The recombinant vector may be introduced into the animal cell, for example, by an electroporation method, a calcium phosphate method or a lipofection method.

[0075] An insect cell such as Sf9 cell, Sf21 cell or the like may also be used as the host. The recombinant vector may be introduced into the insect cell, for example, by a calcium phosphate method, a lipofection method or an electroporation method.

[0076] 4. Production of IP₃ Sponge

[0077] The IP₃ sponge of the present invention may be obtained by culturing the above-described transformant, and recovering the IP₃ sponge from the culture product. The term “culture” as used herein refers to a culture supernatant, a cultured cell or microbial cell, or a cell or microbial cell debris.

[0078] The transformant of the invention is cultured according to a general method employed for culturing the host.

[0079] A medium for culturing the transformant obtained from a microorganism host such as E.coli or yeast may be either a natural or a synthetic medium providing that it contains carbon sources, nitrogen sources, inorganic salts and the like assimilable by the microorganism, and that it can efficiently culture the transformant.

[0080] As carbon sources, carbohydrate such as glucose, fructose, sucrose, starch; organic acids such as acetic acid, propionic acid; and alcohols such as ethanol and propanol may be used.

[0081] As nitrogen sources, ammonia; ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate; other nitrogen-containing compounds; Peptone; meat extract; corn steep liquor and the like may be used.

[0082] As inorganic substances, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, iron(II) sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like may be used.

[0083] The cultivation is generally performed under aerobic conditions such as shaking or aeration agitating conditions at 37° C. for 6 to 24 hours. During the cultivation, pH is maintained at 7.0 to 7.5. pH is regulated with an inorganic or organic acid, an alkali solution or the like. If necessary, an antibiotic such as ampicillin, tetracycline or the like may be added to the medium during the cultivation.

[0084] When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium at need. For example, isopropyl 1-thio-β-D-galactoside (IPTG) may be added to the medium when culturing a microorganism transformed with an expression vector pET-3a having T7 promoter (that is inducible with IPTG). When culturing a microorganism transformed with an expression vector using trp promoter (that is inducible with indole acetic acid (IAA)), IAA may be added to the medium.

[0085] A transformant obtained with an animal cell host may be cultured in a generally used medium such as RPMII640 medium or DMEM medium, or a medium obtained by supplementing the generally used medium with fetal bovine serum and the like.

[0086] The cultivation is generally conducted under 5% CO₂ at 37° C. for 1 to 30 days. If necessary, an antibiotic such as kanamycin, penicillin or the like may be added to the medium during the cultivation.

[0087] After the cultivation, in the case where a microbial cell or a cell intracelluraly produced the IP₃ sponge of the invention, the IP₃ sponge is collected by disrupting the microbial cell or the cell by sonication, freezing and thawing method, or homogenizing. In the case where a microbial cell or a cell extracellularly produced the IP₃ sponge of the invention, the microbial cell or the cell is removed from the culture through centrifugation or the like before, or the culture solution is directly subjected to the isolation/purification procedure. The IP₃ sponge of the invention is isolated and purified from the culture through a general biochemical method for isolating and purifying a protein, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, or a combination thereof.

[0088] 5. Therapeutic Agent and Agent for Gene-Therapy

[0089] Since the protein and the gene of the invention has IP₃ neutralizing activity, they are useful as an antagonist for IP₃-induced calcium, a therapeutic agent and an agent for gene therapy for diseases associated with calcium production. The therapeutic agent or the agent for gene therapy of the invention can be administered orally or parenterally and systemically or locally.

[0090] When the protein or the gene of the invention is used as a therapeutic agent or an agent for gene therapy for disease associated with calcium production, the disease to be treated is not particularly limited. For example, the protein or the gene may be used for diseases in the nervous system, blood vascular system, respiratory system, digestive system, lymphatic system, urinary system, reproduction system or the like for the specific purpose of treatment or prevention. These diseases may be in the form of a single disease or may be complicated by one of these diseases or by some disease other than those mentioned above; any of such forms may be treated with the protein or the gene of the invention.

[0091] When the therapeutic agent of the invention is administered orally, the agent may be formulated into a tablet, capsule, granule, powder, pill, troche, internal liquid agent, suspension, emulsion, syrup or the like. Alternatively, the therapeutic agent may be prepared into a dry product which is re-dissolved just before use. When the therapeutic agent of the invention is administered parenterally, the agent may be formulated into a intravenous injection (including drops), intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, or the like. Injections are supplied in the form of unit dosage ampules or multi-dosage containers.

[0092] These formulations may be prepared by conventional methods using appropriate excipients, fillers, binders, wetting agents, disintegrating agents, lubricating agents, surfactants, dispersants, buffers, preservatives, dissolution aids, antiseptics, flavoring/perfuming agents, analgesics, stabilizers, isotonicity inducing agents, etc. conventionally used in pharmaceutical preparations.

[0093] Each of the above-described formulations may contain pharmaceutically acceptable carriers or additives. Specific examples of such carriers or additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium alginate, water-soluble dextran, sodium carboxymethyl amylose, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerol, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol and lactose. One or a plurality of these additives are selected or combined appropriately depending of the form of the preparation.

[0094] The dosage levels of the therapeutic agent of the invention will vary depending on the age of the subject, the route of administration and the number of times of administration and may be varied in a wide range. When an effective amount of the protein of the invention is administered in combination with an appropriate diluent and a pharmaceutically acceptable carrier, the effective amount of the protein can be in the range from 0.0001 to 1000 mg/kg per administration. The therapeutic agent is administered once a day or in several dosages per day for at least one day.

[0095] When the gene of the invention is used as an agent for gene therapy for diseases associated with calcium production, the gene of the invention may be directly administered by injection. Alternatively, a vector incorporating the gene of the invention may be administered. Specific examples of a suitable vector for this purpose include an adenovirus vector, adeno-associated virus vector, herpes virus vector, vaccinia virus vector and retrovirus vector. The gene of the invention can be administered efficiently by using such a virus vector. Alternatively, the gene of the invention may be enclosed in phospholipid vesicles such as liposomes, and the resultant liposomes may be administered to the subject. Briefly, since liposomes are biodegradable material-containing closed vesicles, the gene of the invention is retained in the internal aqueous layer and the lipid bilayer of liposomes by mixing the gene with the liposomes (a liposome-gene complex). Subsequently, when this complex is cultured with cells, the gene in the complex is taken into the cells (lipofection). Then, the resultant cells may be administered by the methods described below.

[0096] As a method for administering the agent for gene therapy of the invention, local administration to tissues of the central nervous system (brain, spiral cord), blood vascular system (artery, vein, heart), respiratory system (trachea, lung), digestive system (salivary glands, stomach, intestines, liver, pancreas), lymphatic system (lymph node, spleen, thymus), urinary system (kidney), reproduction system (testis, ovary, uterus) or the like may be performed in addition to conventional systemic administration such as intravenous or intra-arterial administration. Further, an administration method combined with catheter techniques and surgical operations may also be employed.

[0097] The dosage levels of the agent for gene therapy of the invention vary depending on the age, sex and conditions of the subject, the route of administration, the number of times of administration, and the type of the formulation. Usually, it is appropriate to administer the gene of the invention in an amount of 0.01-100 mg/adult body/day.

EXAMPLES

[0098] Hereinafter, the present invention will be described in detail by way of examples which do not limit the technical scope of the present invention.

Example 1

[0099] Constuction of Expression Plasmid for High Affinity IP₃-Binding Polypeptide (IP₃ Sponge)

[0100] The N-terminal amino acids (734 amino acids) (polypeptide T734) of a mouse Type-1 IP₃ receptor (mIP₃R1) has a specific IP₃-binding activity. The cDNA portion coding for polypeptide T734 was cloned into E.coli expression vector pET-3a (whose expression is controlled by T7 promoter that is induced upon addition of IPTG) to obtain plasmid pET-T734 (Yoshikawa F. et al., J. Biol. Chem. 271:18277-18284, 1996). Using this plasmid (pET-T734) as a parent plasmid, the following expression plasmids were constructed for IP₃-binding polypeptides. Herein, an IP₃-binding polypeptide with high affinity is also referred to as an “IP₃ sponge”.

[0101] (1-1) Expression Plasmid for High Affinity IP₃ Sponge “T604”

[0102] A gene coding for polypeptide T604 that corresponds to the first methionine (M-1) to the lysine at Position 604 (K-604) of polypeptide T734 was prepared. Specifically, site-directed mutagenesis was conducted by PCR using a complementary oligonucleotide (Yoshikawa F. et al., J Biol Chem, 271:18277-18284, 1996) to introduce a stop codon (TAA) and a subsequent BamHI recognition site (GGATCC) at Position 605 of T734.

[0103] Sense primer: 5′-TGTCAGACATATGCGTGTTGGAA-3′

[0104] NdeI (SEQ ID NO: 5)

[0105] Antisense primer: 5′-CGCGGGATCCTTATTCCGGTTGTTGTGGAGCAGGG-3′

[0106] BamHI (SEQ ID NO: 6)

[0107] The sense primer was introduced with a NdeI cleavage recognition sequence (CATATG) (underlined) including the first methionine codon (ATG).

[0108] A total of 100 μl PCR reaction solution was used. The PCR reaction solution contained 100 ng template DNA, 10 mM KCl, 6 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.2), 2 mM MgCl₂, 0.1% TritonX-100, 10 μg/ml BSA, 200 μM dNTPs, 1 μM sense primer, 1 μM anti-sense primer and 2.5 unit Pfu DNA polymerase. The PCR reaction was performed at 95° C. for 1 min. and then through 30 cycles of: 95° C. for 1 min.; 57° C. for 3 min.; and 72° C. for 3 min.

[0109] The 5′-end of the obtained amplified fragment was treated with NdeI and the 3′-end with BamHI, thereby producing deletion mutant pET-T604 that contains DNA coding for an amino acid sequence corresponding to the amino acid sequence of T734 but with C-terminal deletion up to Position 605.

[0110] (1-2) Expression Plasmid for High Affinity IP₃ Sponge “G224”

[0111] First, a gene coding for polypeptide T604 that corresponds to the first methionine (M-1) to the lysine at Position 604 (K-604) of polypeptide T734 was prepared. Specifically, site-directed mutagenesis was conducted by PCR using a complementary oligonucleotide (Yoshikawa F. et al., J Biol Chem, 271:18277-18284, 1996) to introduce a stop codon (TAA) and a subsequent EcORI recognition site (GAATCC) at Position 605 of T734.

[0112] Sense primer: 5′-TGTCAGA CATATGCGTGTTGGAA-3′

[0113] NdeI (SEQ ID NO: 5)

[0114] Antisense primer: 5′-CCGGAATTCTATTTCCGGTTGTTGTGGAGCAGGG-3′

[0115] EcoRI (SEQ ID NO: 7)

[0116] The PCR was conducted under the same conditions as described in (1-1) above.

[0117] The 5′-end of the thus-obtained amplified fragment was treated with NdeI and the 3′-end with EcoRI, thereby producing deletion mutant pET-T604e that contains DNA coding for an amino acid sequence corresponding to the amino acid sequence of T734 but with C-terminal deletion up to Position 605.

[0118] Then, using deletion mutant pET-T604e as a template, site-directed mutagenesis was performed to introduce BamHI recognition site (GGATCC) immediately before the methionine at Position 224 of polypeptide T604.

[0119] Antisense primer: 5′-CCGGAATTCTTATTTCCGGTTGTTGTGGAGCAGGG-3′

[0120] EcoRI (SEQ ID NO: 7)

[0121] Sense primer: 5′-CGCGGATCCATGAAATGGAGTGATAACAAAGACGACA-3′BamHI (SEQ ID NO: 8)

[0122] The PCR was conducted under the same conditions as described in (1-1) above.

[0123] The thus-obtained amplified fragment (plasmid introduced with mutation) was cleaved with BamHI and EcoRI, thereby obtaining a CDNA fragment coding for Amino acids 224-604. This cDNA fragment was ligated to BamHI-EcoRI site of GST fusion plasmid (pGEX-2T) without a frameshift (in-frame), thereby obtaining plasmid pGEX-G224. Plasmid pGEX-G224 expresses fusion polypeptide G224 (FIG. 1) that includes polypeptide GST and subsequent polypeptide M-224 to K-604.

[0124] (1-3) Expression Plasmid for Low Affinity IP₃-binding Polypeptide

[0125] Site-directed mutagenesis was conducted by sequential PCR using pGEX-G224 as a template.

[0126] The following two mismatched oligonucleotides were synthesized to introduce mutation (K508A) at Position 508 of T604 where alanine was substituted for lysine (K-508): 5′-GAGAGCGGCAGGCACTGATGAGGG-3′ (SEQ ID NO:9) 5′-CCCTCATCAGTGCCTGCCGCTCTC-3′ (SEQ ID NO:10)

[0127] Using the above primers, site-directed mutagenesis was conducted by sequential PCR. The PCR conditions and the composition of the reaction solution were as follows:

[0128] Primary reaction 1

[0129] Sense primer: 5′-GAGAGCGGCAGGCAACTGATGAGGG-3′(SEQ ID NO: 9)

[0130] Antisense primer: 5′-CCGGAATTATTATTCCGGTTGTTGTGGAGCAGGG-3′EcoRI (SEQ ID NO: 7)

[0131] The PCR was conducted under the same conditions as described in (1-1) above.

[0132] Primary reaction 2

[0133] Sense primer: 5′-CGCGGATCCATGAAAATGGAGTGATAACAAAGACGACA-3′

[0134] BamHI (SEQ ID NO: 8)

[0135] Antisense primer: 5′-CCCTCATCAGTGCCTGCCGCTCTC-3′ (SEQ ID NO: 10)

[0136] The PCR was conducted under the same conditions as described in (1-1) above.

[0137] Secondary reaction

[0138] Ten μl of the PCR reaction product resulting through Primary reactions 1 and 2, and 1 μM each of primers (SEQ ID NOS: 7 and 8) were used to conduct PCR under the same conditions as the primary reactions.

[0139] The obtained amplified fragment was cleaved with BamHI and EcoRI. The cleaved fragment was ligated to BamHI-EcoRI site of GST fusion plasmid pGEX-2T without a frameshift (in frame), thereby obtaining plasmid pGEX-G224-m30. This mutant plasmid expresses polypeptide G224-m30 having the point mutation K508A (FIG. 1, m30).

[0140] (1-4) Expression Plasmid for High Affinity IP₃ Sponge “G224-m49”

[0141] Using pGEX-G224 as a template, site-directed mutagenesis was conducted by sequential PCR.

[0142] The following two mismatched oligonucleotides were synthesized to introduce a mutation (R441Q) at Position 441 of T604 where glutamine was substituted for alginine (R-441):

[0143] 5′-GCTGAGGTTCAAGACCTGGACTTTG-3′ (SEQ ID NO: 11)

[0144] 5′-AAAGTCCAGGTCTTGAAGAACCTCAGC-3′ (SEQ ID NO: 12)

[0145] Primary reaction 1

[0146] Sense primer: 5′-GCTGAGGTTCAAGACCTGGACTTTG-3′ (SEQ ID NO: 11)

[0147] Antisense primer: 5′-CCGGAATTCCTTAATTTCCGGTTGTTGTGGAGCAGGG-3′

[0148] EcoRI (SEQ ID NO: 7)

[0149] The PCR was conducted under the same conditions as described in (1-3) above.

[0150] Primary reaction 2

[0151] Sense primer: 5′-CGCGGATCCATGAAATGGAGTGATAACAAAGACGACA-3′

[0152] BamHI (SEQ ID NO: 8)

[0153] Antisense primer: 5′-AAAGTCCAGGTCTTGAACCTCAGC-3′ (SEQ ID NO: 12)

[0154] The PCR was conducted under the same conditions as described in (1-3) above.

[0155] Secondary reaction

[0156] Ten μl of the PCR reaction product resulting through Primary reactions 1 and 2, and 1 μM each of primers (SEQ ID NOS: 6 and 8) were used to conduct PCR under the same conditions as those of the primary reactions.

[0157] The obtained amplified fragment was cleaved at a BamHI-EcoRI site. The cleaved fragment was ligated to BamHI-EcoRI site of GST fusion plasmid pGEX-2T without a frameshift (in frame), thereby obtaining plasmid pGEX-G224-m49. This mutant plasmid expresses polypeptide G224-m49 having the point mutation R441Q (FIG. 1, m49).

Example 2 Expression and Preparation of High Affinity IP₃-Binding Polypeptide with E.coli

[0158] Since the IP₃-binding core mostly results in insoluble inclusion bodies, the expression amount is low. Thus, the present inventors have modified the IP₃-binding region through gene engineering to produce a high affinity IP₃-binding polypeptide which is of lower molecule, which is capable of stable mass-expression, which can be recovered as a soluble protein, which has a higher affinity, and which has as high specificity as a conventional IP₃ receptor.

[0159] By low-temperature cultivation (16-22° C.), polypeptide T734 can be mass-expressed in a stable manner with a relatively high soluble fraction recovery (Kd=50±2.4 nM, B_(max)=46 pmol/mg protein, 1.85 mg/l E.coli culture (corresponding to about 0.5 g of wet E.coli)). However, the inclusion bodies amount to more than ten times the amount of the soluble fraction (Yoshikawa F. et al., J. Biol Chem. 271: 18277-18284, 1996).

[0160] First of all, smaller polypeptides that had the above-described characteristics were prepared.

[0161] The pET-type and pGEX-type expression plasmids obtained in Example 1 were introduced into E.coli BL21 (DE3) and JM109, respectively, by transformation method. Expression induction with IPTG and preparation of expression proteins from E.coli were mainly conducted by modifying the method of Yoshikawa et al (Yoshikawa F. et al., J. Biol Chem. 271: 18277-18284, 1996).

[0162] Specifically, E.coli introduced with respective plasmids were shake cultured in L broths (containing 100 μg/ml ampicillin) at 22° C. When the absorption OD₆₀₀ became about 1.5, IPTG was added to 0.5 mM. After a few hours of shake culture at 16° C., each of the E.coli was recovered through centrifugation and suspended in PBS containing protease inhibitors. (1 mM PMSF, 10 μM leupeptin, 1 μM pepstatin A, 2 μg/ml aprotinin). Each of the E.coli was disrupted by sonication. Then, each supernatant containing the expression polypeptide (soluble fraction) was collected by ultracentrifugation (Beckman Ti35 rotor, 25,000 rpm, 1 hr., 4° C.).

[0163] GST fusion polypeptides were purified from the soluble fractions by affinity purification using Glutathione-Sepharose column (Pharmacia LKB). Specifically, each of the GST fusion polypeptides was eluted from the column with 10 mM glutathione/50 mM Tris-HCl (pH 8.0) by mainly following the manual provided by the manufacturer. The polypeptide solutions were equilibrated with 10 mM HEPES-KOH (pH 7.2), 88 mM NaCl and 1 mM KCl using PD10 desalted column (Pharmacia LKB), and then dispensed, thereby obtaining the IP₃ sponges (FIG. 1: G224, m30, m49 and GST). The IP₃ sponges were stored at −80° C. until they were used.

[0164] A series of deletion mutants based on polypeptide T734 were prepared by serially shortening the length of the polypeptide T734 from the C-terminus. The analysis of the deletion mutants indicated that T705 and T699 had no marked characteristic difference with T734. In the cases of polypeptides T569, T572 and T576, the expression amounts of the soluble proteins were lower than T734. Stable mass-expression of soluble protein was successful with polypeptide T604 which was obtained by deleting the C-terminus of T734 up to Amino acid 605 (FIG. 1).

[0165] With reference to FIG. 1, the uppermost (IP₃R1) is the N-terminal amino acids of the IP₃ receptor including the IP₃-binding core region (core: Amino acids 226-578). T604 (Amino acids 1-604), G224 (GST+Amino acids 224-604), G224m30 (G224 introduced with K508A mutation), G224m49 (G224 introduced with R441Q mutation), and GST (derived from pGEX-2T)) are also shown in FIG. 1.

[0166] T604 had a [³H]IP₃-binding activity substantially equivalent to that of T734 (Kd=45 nM), and a higher yield of soluble protein (B_(max)=690 pmol/mg protein). Specifically, the yield was 19 mg/l E.coli culture (FIGS. 2B and 2C, Table 1). TABLE 1 Expression of IP₃-binding site in E. coli Expression B_(max) [pmol/μg efficiency (mg/l Kd purified Protein E. coli culture) [nM] protein] Purified IP₃R^(a) — 83 2.1 T734^(b) 1.85 (50)^(c) ND T604 19 7.6/(45)^(c) ND G224 30 0.083 1.6 G224m49 ND 0.043 1.7 G224m30 ND 330 3.0

[0167] The total expression amount of polypeptide T604 substantially equaled to that of polypeptide T734 but T604 had a remarkably improved soluble protein yield. The yield of soluble protein of polypeptide T604 was substantially the same at 30° C. and 37° C., and reached the peak within 2 hours after initiating expression induction.

[0168]FIG. 2A shows the result of Western blotting analysis of the protein (0.1 μg) obtained from an E.coli extract solution (soluble fraction) that expresses polypeptide T604 (66 kDa). As a control, a cell extract solution obtained by transforming a vector that does not include T604 (pET-3a) was used. FIG. 2B shows a comparison of the total amounts of specific IP₃-binding contained in 0.7 μl soluble fractions, for T734, T604, and the control vector. FIG. 2C shows the result of Schatchard plot analysis where the binding between 3 μg of T604 soluble fraction and 9.6 nM [³H]IP₃ was competitively inhibited with non-labeled IP₃ (cold IP₃) at various concentrations. The results were Kd=45±7.6, B_(max)=690±64 pmol/mg protein.

[0169] When T734 was serially deleted from the N-terminus, a very short N-terminal deletion of T734 (e.g., a deletion of 31 amino acids) caused lack of IP₃-binding activity even the deletion was outside the core region. However, the polypeptide retrieved the IP₃-binding activity when the N-terminus was deleted to Amino acid 220-225, near the N-terminus of the core region (Yoshikawa et al, 1996). The theory for this is unknown, but presumably, the formation of the three-dimensional structure for active core region is somehow interrupted depending on the degree of deletion. Although the active polypeptide with the N-terminal deletion up to Amino acid 220-225 had a relatively high affinity, the amount of soluble protein expressed was lower.

[0170] As described above, a protein obtained by deleting Amino acids 1-223 of polypeptide T604 (N4-T604; Amino acids 224-604) had a higher activity (about 3 times high) but lower production than those of the original T604. Accordingly, polypeptide T604 seemed to be the most suitable polypeptide for stably mass-expressing only the high affinity IP₃-binding region as a soluble protein.

Example 3 Expression of IP₃ Sponge

[0171] (i) [³H]-IP₃-binding Inhibition Experiment

[0172] Based on the results obtained in Example 2, an IP₃-binding polypeptide with a higher affinity was produced. As described above, when the amino-terminal Amino acids 1-223 of polypeptides T604 and T734 were deleted, high [³H]IP₃-binding activities were obtained. Even Amino acid region 224-579 (a polypeptide that almost corresponds to the core region) consisting of only 356 amino acid residues has an affinity as high as Kd=2.3 nM (Yoshikawa et al., 1996, supra). However, as described above, these polypeptides have lower soluble protein expression levels. In other words, longer amino terminal deletion may result in a higher affinity on one hand, but it also lowers the expression amount and expression stability of soluble proteins by rendering most of proteins as insoluble inclusion bodies.

[0173] In general, stability, solubility and an expression level of a foreign polypeptide are known to be improved when it is made into a GST fusion body. In this example, fusion proteins G224, G224-m30 and G224-m49 consisting of GST and an IP₃-binding site (Amino acid region 224-604) were prepared by ligating GST to replace the N-terminal region (Amino acids 1-223) of the IP₃ receptor (FIG. 1).

[0174] The IP₃-binding activities of these fusion proteins were measured mainly by the method of Yoshikawa et al (1996).

[0175] Each fusion protein (IP₃ sponge) (0.2 μg) was mixed with 100 μl of binding buffer-α (50 mM Tris-HCl (pH 8.0 at 4° C.), 1 mM EDTA, 1 mM β-mercaptoethanol) that contained 9.6 nM D-myo-[³H](1,4,5)IP₃ (777 GBq/mmol; DuPont NEN) (hereinafter, abbreviated as “[³H]IP₃”) and various concentrations of non-labeled D-myo-(1,4,5)IP₃ (Dojindo) (hereinafter, abbreviated as “cold IP₃”). The mixture was left to stand on ice for 10 minutes. To the mixture, 4 μl of 50 mg/ml γ-globulin (Sigma) (final concentration: 1 mg/ml) and 100 μl of 30% PEG 6000 (Sigma)/binding buffer-a solution (final concentration: 15%) were added. The resultant mixture was left to stand on ice for 5 minutes, and then centrifuged at 10,000×g at 2° C. for 5 minutes to collect polypeptide/PEG complex. PEG-precipitated [³H]IP₃-binding polypeptide was well solubilized with 180 μl solubilizer Solvable (DuPont NEN). The resultant was neutralized with 18 μl glacial acetic acid and then added to 5 ml liquid scintillation counter (Atomlight [DuPont NEN]) to measure the radioactivity (first radioactivity). Non-specific binding of each protein was determined by measuring the second radioactivity in the presence of 2 μM or 10 μM cold IP₃. Then, a specific binding value of each protein was obtained by subtracting the second radioactivity (non-specific binding value) from the first radioactivity values.

[0176] Scatchard plot analysis was conducted under the following conditions. For low-affinity polypetides (G224-m30 and control GST), the binding experiment was conducted in 100 μl binding buffer a by adding 9.6 nM [³H]IP₃ (DuPont NEN) and 10-20 nM of cold IP₃ to 2 μg of IP₃-binding polypeptide, and by adding 9.6 nM [³H]IP₃ (DuPont NEN) and 50 nM-2 μM of cold IP₃ to 0.01 μg IP₃-binding polypeptide. For high-affinity IP₃ sponges (G224 and G224-m49), binding experiment was conducted with 0.02 μg IP₃ sponges at [³H]-IP₃ concentrations of 0.15, 0.3, 0.6, 1.2, 2.4, 4.8 and 9.6 nM without adding cold IP₃.

[0177] The inhibition effects of the IP₃-binding polypeptides (IP₃ sponges) on [H]IP₃-binding activity of cerebellar microsome was analyzed as follows.

[0178] A microsomal fraction was prepared from the cerebella of mice ddY (Nippon SLC) mainly by following the method of Nakada et al. (Nakada S. et al., Biochem. J. 277:125-131, 1991). In 100 μl of binding buffer α, various concentrations of the IP₃ sponges were added respectively to see the changes in the binding between the cerebellar microsome (40 μg) and 9.6 nM [³H]-IP₃ according to the above method (see Scatchard plot analysis).

[0179] As a result, the affinity of polypeptide G224 was found out to be 500 times higher than that of polypeptide T734 (Kd=83 pM, B_(max)=1.6 pmol/μg protein) (FIG. 3A). Polypeptide G224 binds well to (1,4,5)IP₃ and (2,4,5)IP₃ and the yield of IP₃-binding protein was about 30 mg/l E.coli culture (Table 1). After purifying the protein with a glutathione column and a subsequent PD10 column, the yield was about 24 mg/l. The binding activity was augmented when R441Q mutation was introduced into polypeptide T734 (Yoshikawa et al., 1996, supra). The affinity of polypeptide G224-m49 (G224 introduced with R441Q mutation) doubled and became about 1,000 times higher than that of polypeptide T734 (Kd=about 43 pM, B_(max)=1.7 pmol/μg protein) (FIG. 3B, Table 1). The binding activity decreased when polypeptide T734 was introduced with K508A mutation (Yoshikawa et al., 1996 (supra)). Similarly, the binding activity of polypeptide G224-m30 decreased when G224 was introduced with K508A mutation and became as low as about 1/4,000 of polypeptide G224 and about 1/7,700 of polypeptide G224-m49 (Kd=about 330 nM, B_(max)=3.0 pmol/μg protein) (FIG. 3C, Table 1).

[0180] (ii) IP₃-binding Inhibition via Absorption by Novel IP₃ Sponge

[0181] IP₃-binding polypeptides G224 and G224-m49 have powerful IP₃-binding activities that are 500 to 1,000 times higher than that of the original IP₃ receptor. Polypeptides G224 and G224-m49 were tested for their use as an IP₃-specific absorption body (sponge) (IP₃ sponge), i.e., whether they can decrease the amount of IP₃-binding by the IP₃ receptors in a solution by competitively absorbing IP₃ in the solution (FIG. 4).

[0182] Mouse cerebellum is a tissue that is rich in IP₃ receptor and whose microsomal fraction has a [³H]IP₃-binding activity which is at least 50 times higher than those in other tissues (Maeda et al., 1990 (supra)). Binding between 40 μg cerebellar microsome (Kd=21 nM, B_(max)=23 pmol/mg protein) and 9.6 nM [³H]IP₃ in 100 μl solution was analyzed for percentage (%) of competitive inhibition at various concentrations of IP₃ sponges where the activity under the absence of IP₃ sponge was considered 100%. It was calculated that, there were about 0.92 pmol of IP₃-binding site of cerebellum IP₃ receptor and 0.96 pmol of [³H]IP₃ present in the 100 μl solution.

[0183] As a result, no inhibition effect was observed for control GST even when the IP₃ sponge concentration was 100 μg/ml (FIG. 4). On the other hand, for high-affinity polypeptides G224 and G224-m49, strong IP₃-binding inhibition activities were observed and IC₅₀ was about 10 μg/ml (FIG. 4). Polypeptide G224-m30 with low affinity had low inhibition activity with IC₅₀ of 100 μg/ml. According to this in vitro experiment system, the IP₃ sponge tended to precipitate with the microsome membrane when the IP₃ sponge concentration exceeded about 25 μg/ml, and so the concentration-dependent curves were likely to fluctuate (FIG. 4). Thus, the apparent inhibition of G224-m30 observed at IP₃ sponge concentration exceeding 25 μg/ml could be due to precipitation under high concentration.

[0184] These results show that [³H]IP₃-binding of the IP₃ receptor can efficiently be inhibited according to the binding affinity and the concentration of the IP₃ sponge used. High affinity IP₃-binding polypeptide of the invention is a novel IP₃ sponge that can be used as an IP₃ neutralizing agent, or an antagonist for IP₃-induced calcium.

Example 4 Test of Inhibiting IP₃-Induced Ca²⁺ Release (IICR)

[0185] To conduct a test of inhibiting IP₃-induced Ca²⁺ release, a microsomal fraction was prepared from mouse cerebellum as described in Example 3. The fraction was suspended in Buffer B, dispensed, and stored at −80° C. until it was used.

[0186] Composition of Buffer B was 110 mM KCl, 10 mM NaCl, 5 mM KH₂PO₄, 1 mM DDT, and 50 mM HEPES-KOH (pH 7.2) (containing a cocktail of protease inhibitors [0.1 mM PMSF, 10 μM leupeptin, 10 μM pepstatin A, 10 μM E-64] and 2 mM MgCl₂).

[0187] An IP₃-induced Ca²⁺ release activity of cerebellar microsome was determined by using fura-2 (Molecular Probe) as a fluorescent Ca²⁺ indicator. Specifically, excitations upon addition of IP₃ at two wavelengths (340 nm and 380 nm) were measured with fluorescence spectrophotometer CAF110 (Nihon Bunko) to see the change in the fluorescent intensity ratio (F340/F380) at 500 nm.

[0188] IP₃-induced Ca²⁺ release from the cerebellar microsome is generally EC₅₀=100-200 nM IP₃. Cerebellar microsome (100 μg) was mixed with 500 μl of a release buffer (Buffer B containing 1 mM MgCl₂, 2 μM fura-2, 1 mM DTT, 10 mM creatine phosphate, 40 U/ml creatine kinase, 1 μg/ml oligomycin, and the cocktail of protease inhibitors) in a measurement cuvette with a stirrer bar. The following reaction was conducted at 30° C. while constantly stirring with the stirrer bar.

[0189] One mM of ATP was added to the mixture in the cuvette to activate Ca²⁺ pumping (Ca²⁺-ATPase), whereby Ca²⁺ was incorporated into the inner space of microsome (Ca²⁺ loading). Ca²⁺ loading was confirmed by monitoring until the decrease of fura-2 fluorescent level became constant. The change in the fura-2 fluorescent intensity ratio was measured (F340/F380) at a subthreshold level.

[0190] The effect of IP₃ sponge on inhibiting IP₃-induced Ca²⁺ release activity of cerebellar microsome was analyzed as follows. After the addition of ATP, the curve of fura-2 fluorescent intensity was monitored until the decrease became constant. Then, various concentrations of IP₃ sponges were added. After 1 min., 50 nM to 1 μM of IP₃ was added to the reaction mixture to observe the change of fura-2 fluorescent intensity induced by the IP₃.

[0191] The IP₃ sponge concentration dependency was determined as follows. High affinity polypeptide G224 of 3.125, 6.25, 12.5, 25, 50, 100, 200 μg/ml were added to the reaction mixture, respectively. After about 1 min., 100 nM of IP₃ was added to measure the Ca²⁺ release activity induced by the IP₃. The concentration dependency of low affinity polypeptide G224-m30 was determined by adding G224-m30 of 200, 400 and 500 μg/ml. After about 1 min., 100 nM of IP₃ was added to measure the Ca²⁺ release activity induced by the IP₃. In addition, G224-m30 of 500 μg/ml was also added, and after about 1 min., 50 nM of IP₃ was added to measure the Ca²⁺ release activity induced by the IP₃.

[0192] As a result, it was found that the IP₃ sponges specifically inhibited in a competitive manner the IP₃-binding by the IP₃ receptor of cerebellar microsome by absorbing the IP₃ (FIGS. 5A-5F, 6A-6G and 7). In FIGS. 5A-5F and 6A-6G, the vertical axis represent the change in fura-2 fluorescent intensity ratio (F340/F380) (i.e., change in the amount of Ca²⁺), and the horizontal axis represents the time (sec).

[0193] As shown in FIGS. 5A, 5B and 5C, in the absence of IP₃ sponge (controls), IP₃-induced Ca²⁺ release activities were dependent on IP₃ concentration. Low-affinity polypeptide G224-m30 at a concentration of 500 μg/ml had no inhibiting effect on Ca²⁺ release with 100 nM IP₃ (FIG. 5E). Little difference was found between G224-m30 and the control for effects on inhibiting 50 nM IP₃ (FIG. 5E). With GST only, even at a high concentration of 632 μg/ml, no change was seen in Ca²⁺ release activity induced with 50 nM IP₃ (FIG. 5F). Thus, in each case, no marked difference was noted with the control.

[0194] On the contrary to the above results, high affinity polypeptide G224 had a significant inhibition effect on IP₃-induced Ca²⁺ release depending on its concentration (FIGS. 6A-6G). The high affinity polypeptide G224 had the greatest inhibition effect at 100 μg/ml and almost completely inhibited the IP₃-induced Ca²⁺ release (FIG. 6F).

[0195] The peak values of Ca²⁺ release obtained by adding G224 at each concentration shown in FIGS. 6A-6G, were plotted where the peak obtained in the absence of G224 was considered 100% (FIG. 7). The horizontal axis represents each concentration of polypeptide G224 and the verticle axis represents the peak value of Ca²⁺ release. As can be appreciated from FIG. 7, the concentration of polypeptide G224 required for 50% inhibition of IP₃-induced Ca²⁺ release was about 20 μg/ml.

[0196] Accordingly, it was found that the high affinity IP₃-binding polypeptide acted as an IP₃ sponge and specifically inhibited, in a concentration-dependant manner, the IP₃-induced Ca²⁺ release by the IP₃ receptor on cerebellar microsome.

[0197] The present invention provides a polypeptide having a high affinity binding activity to inositol 1,4,5-trisphosphate, a gene encoding the polypeptide, a recombinant vector including the gene, a transformant including the vector and a method for producing the polypeptide.

[0198] The polypeptide of the invention can be used to control the inhibition of a specific cell function that depends on an IP₃-induced calcium signal transmission (IP₃ neutralizing agent, antagonist for IP₃-induced calcium, etc.). Furthermore, the polypeptide and the gene of the present invention is useful as an IP₃ signal detecting agent for inhibiting activation of IP₃-induced calcium signal trasmission. The gene of the invention is also useful as a therapeutic agent for treating a disease associated with calcium production.

[0199] Various other modifications will be apparent to and can be readily made by those skilled in the art without departing from the scope and spirit of the claims appended hereto be limited to the description as set forth herein, but rather that the claims be broadly construed.

[0200] All publications, patents, and patent applications cited herein are incorperated herein by reference in their entirety.

[0201] The following are information on sequences described herein:

1 14 1 1812 DNA Mus musculus CDS (1)..(1812) 1 atg tct gac aaa atg tcg agt ttc cta cat att gga gac att tgt tct 48 Met Ser Asp Lys Met Ser Ser Phe Leu His Ile Gly Asp Ile Cys Ser 1 5 10 15 ctg tat gcg gag gga tct acg aat gga ttt atc agc acc tta ggc ttg 96 Leu Tyr Ala Glu Gly Ser Thr Asn Gly Phe Ile Ser Thr Leu Gly Leu 20 25 30 gtt gat gac cgt tgt gtt gta cag cca gaa gcc ggg gac ctt aac aat 144 Val Asp Asp Arg Cys Val Val Gln Pro Glu Ala Gly Asp Leu Asn Asn 35 40 45 cca ccc aag aaa ttc aga gac tgc ctc ttt aag cta tgt cct atg aat 192 Pro Pro Lys Lys Phe Arg Asp Cys Leu Phe Lys Leu Cys Pro Met Asn 50 55 60 cga tac tcc gca cag aaa cag ttc tgg aaa gct gct aag ccc ggg gcc 240 Arg Tyr Ser Ala Gln Lys Gln Phe Trp Lys Ala Ala Lys Pro Gly Ala 65 70 75 80 aac agc act aca gat gca gtg ctg ctc aac aaa ttg cat cat gct gca 288 Asn Ser Thr Thr Asp Ala Val Leu Leu Asn Lys Leu His His Ala Ala 85 90 95 gac ttg gaa aag aag cag aat gag aca gaa aac agg aaa ttg ttg ggg 336 Asp Leu Glu Lys Lys Gln Asn Glu Thr Glu Asn Arg Lys Leu Leu Gly 100 105 110 acc gtc atc caa tat ggc aac gtg atc cag ctc ctg cat ttg aaa agc 384 Thr Val Ile Gln Tyr Gly Asn Val Ile Gln Leu Leu His Leu Lys Ser 115 120 125 aat aaa tac ctg act gtg aat aag agg ctc cca gcc ttg cta gag aag 432 Asn Lys Tyr Leu Thr Val Asn Lys Arg Leu Pro Ala Leu Leu Glu Lys 130 135 140 aat gcc atg agg gtg acg ttg gac gag gct gga aat gaa ggg tcc tgg 480 Asn Ala Met Arg Val Thr Leu Asp Glu Ala Gly Asn Glu Gly Ser Trp 145 150 155 160 ttt tac att caa cca ttt tac aag ctt cgc tcc atc gga gac agt gtg 528 Phe Tyr Ile Gln Pro Phe Tyr Lys Leu Arg Ser Ile Gly Asp Ser Val 165 170 175 gtc ata ggc gac aag gta gtt ttg aat cct gtc aat gct ggc cag cct 576 Val Ile Gly Asp Lys Val Val Leu Asn Pro Val Asn Ala Gly Gln Pro 180 185 190 cta cat gcc agc agt cat cag ctg gtg gat aac cca ggc tgc aat gag 624 Leu His Ala Ser Ser His Gln Leu Val Asp Asn Pro Gly Cys Asn Glu 195 200 205 gtc aac tcc gtc aac tgt aat aca agc tgg aag ata gtg ctt ttc atg 672 Val Asn Ser Val Asn Cys Asn Thr Ser Trp Lys Ile Val Leu Phe Met 210 215 220 aaa tgg agt gat aac aaa gac gac att ctc aaa gga ggt gat gtg gtg 720 Lys Trp Ser Asp Asn Lys Asp Asp Ile Leu Lys Gly Gly Asp Val Val 225 230 235 240 agg ctc ttc cat gcc gag caa gag aag ttt ctc acc tgt gat gag cac 768 Arg Leu Phe His Ala Glu Gln Glu Lys Phe Leu Thr Cys Asp Glu His 245 250 255 cgg aag aag cag cat gtg ttc ctg agg acc acc ggc agg cag tca gcc 816 Arg Lys Lys Gln His Val Phe Leu Arg Thr Thr Gly Arg Gln Ser Ala 260 265 270 acg tcg gcc acc agt tct aaa gcc ctg tgg gaa gtg gag gta gtc cag 864 Thr Ser Ala Thr Ser Ser Lys Ala Leu Trp Glu Val Glu Val Val Gln 275 280 285 cac gac cca tgt cgg ggt gga gct ggg tac tgg aat agc ctc ttc cgg 912 His Asp Pro Cys Arg Gly Gly Ala Gly Tyr Trp Asn Ser Leu Phe Arg 290 295 300 ttc aag cac ctg gct aca ggg cat tac ttg gct gca gag gta gac cct 960 Phe Lys His Leu Ala Thr Gly His Tyr Leu Ala Ala Glu Val Asp Pro 305 310 315 320 gac ttt gag gaa gaa tgc ctg gag ttt cag ccc tca gtg gac cct gat 1008 Asp Phe Glu Glu Glu Cys Leu Glu Phe Gln Pro Ser Val Asp Pro Asp 325 330 335 cag gat gca tct cgg agt agg ttg aga aac gcg caa gaa aaa atg gta 1056 Gln Asp Ala Ser Arg Ser Arg Leu Arg Asn Ala Gln Glu Lys Met Val 340 345 350 tac tct ctg gtc tcc gtg cct gaa ggc aac gac atc tcc tcc atc ttt 1104 Tyr Ser Leu Val Ser Val Pro Glu Gly Asn Asp Ile Ser Ser Ile Phe 355 360 365 gag cta gac ccc acg act ctg cgt gga ggt gac agc ctt gtc cca agg 1152 Glu Leu Asp Pro Thr Thr Leu Arg Gly Gly Asp Ser Leu Val Pro Arg 370 375 380 aac tcc tat gtc cgt ctc aga cac ctg tgc acc aac acc tgg gta cac 1200 Asn Ser Tyr Val Arg Leu Arg His Leu Cys Thr Asn Thr Trp Val His 385 390 395 400 agc aca aac atc ccc atc gac aag gaa gag gag aag cct gtg atg ctg 1248 Ser Thr Asn Ile Pro Ile Asp Lys Glu Glu Glu Lys Pro Val Met Leu 405 410 415 aaa att ggt acc tct ccc ctg aag gag gac aag gaa gca ttt gcc ata 1296 Lys Ile Gly Thr Ser Pro Leu Lys Glu Asp Lys Glu Ala Phe Ala Ile 420 425 430 gtt cct gtt tcc cct gct gag gtt cgg gac ctg gac ttt gcc aat gat 1344 Val Pro Val Ser Pro Ala Glu Val Arg Asp Leu Asp Phe Ala Asn Asp 435 440 445 gcc agc aag gtg ctg ggc tcc atc gct ggg aag ttg gaa aag ggc acc 1392 Ala Ser Lys Val Leu Gly Ser Ile Ala Gly Lys Leu Glu Lys Gly Thr 450 455 460 atc acc cag aat gag aga agg tct gtc acg aag ctt ttg gaa gac ttg 1440 Ile Thr Gln Asn Glu Arg Arg Ser Val Thr Lys Leu Leu Glu Asp Leu 465 470 475 480 gtt tac ttt gtc acg ggt gga act aac tct ggc caa gac gtg ctt gaa 1488 Val Tyr Phe Val Thr Gly Gly Thr Asn Ser Gly Gln Asp Val Leu Glu 485 490 495 gtt gtc ttc tct aag ccc aat cga gag cgg cag aag ctg atg agg gaa 1536 Val Val Phe Ser Lys Pro Asn Arg Glu Arg Gln Lys Leu Met Arg Glu 500 505 510 cag aat att ctc aag cag atc ttc aag ctg ttg cag gcc ccc ttc acg 1584 Gln Asn Ile Leu Lys Gln Ile Phe Lys Leu Leu Gln Ala Pro Phe Thr 515 520 525 gac tgc ggg gat ggc ccg atg ctt cgg ctg gag gag ctg ggg gat cag 1632 Asp Cys Gly Asp Gly Pro Met Leu Arg Leu Glu Glu Leu Gly Asp Gln 530 535 540 cgc cat gct cct ttc aga cat att tgc cga ctc tgc tac agg gtc ctg 1680 Arg His Ala Pro Phe Arg His Ile Cys Arg Leu Cys Tyr Arg Val Leu 545 550 555 560 cga cac tca cag caa gac tac agg aag aac cag gag tac ata gcc aag 1728 Arg His Ser Gln Gln Asp Tyr Arg Lys Asn Gln Glu Tyr Ile Ala Lys 565 570 575 cag ttt ggc ttc atg cag aag cag att ggc tat gac gtg ctg gcc gaa 1776 Gln Phe Gly Phe Met Gln Lys Gln Ile Gly Tyr Asp Val Leu Ala Glu 580 585 590 gac acc atc act gcc ctg ctc cac aac aac cgg aaa 1812 Asp Thr Ile Thr Ala Leu Leu His Asn Asn Arg Lys 595 600 2 604 PRT Mus musculus 2 Met Ser Asp Lys Met Ser Ser Phe Leu His Ile Gly Asp Ile Cys Ser 1 5 10 15 Leu Tyr Ala Glu Gly Ser Thr Asn Gly Phe Ile Ser Thr Leu Gly Leu 20 25 30 Val Asp Asp Arg Cys Val Val Gln Pro Glu Ala Gly Asp Leu Asn Asn 35 40 45 Pro Pro Lys Lys Phe Arg Asp Cys Leu Phe Lys Leu Cys Pro Met Asn 50 55 60 Arg Tyr Ser Ala Gln Lys Gln Phe Trp Lys Ala Ala Lys Pro Gly Ala 65 70 75 80 Asn Ser Thr Thr Asp Ala Val Leu Leu Asn Lys Leu His His Ala Ala 85 90 95 Asp Leu Glu Lys Lys Gln Asn Glu Thr Glu Asn Arg Lys Leu Leu Gly 100 105 110 Thr Val Ile Gln Tyr Gly Asn Val Ile Gln Leu Leu His Leu Lys Ser 115 120 125 Asn Lys Tyr Leu Thr Val Asn Lys Arg Leu Pro Ala Leu Leu Glu Lys 130 135 140 Asn Ala Met Arg Val Thr Leu Asp Glu Ala Gly Asn Glu Gly Ser Trp 145 150 155 160 Phe Tyr Ile Gln Pro Phe Tyr Lys Leu Arg Ser Ile Gly Asp Ser Val 165 170 175 Val Ile Gly Asp Lys Val Val Leu Asn Pro Val Asn Ala Gly Gln Pro 180 185 190 Leu His Ala Ser Ser His Gln Leu Val Asp Asn Pro Gly Cys Asn Glu 195 200 205 Val Asn Ser Val Asn Cys Asn Thr Ser Trp Lys Ile Val Leu Phe Met 210 215 220 Lys Trp Ser Asp Asn Lys Asp Asp Ile Leu Lys Gly Gly Asp Val Val 225 230 235 240 Arg Leu Phe His Ala Glu Gln Glu Lys Phe Leu Thr Cys Asp Glu His 245 250 255 Arg Lys Lys Gln His Val Phe Leu Arg Thr Thr Gly Arg Gln Ser Ala 260 265 270 Thr Ser Ala Thr Ser Ser Lys Ala Leu Trp Glu Val Glu Val Val Gln 275 280 285 His Asp Pro Cys Arg Gly Gly Ala Gly Tyr Trp Asn Ser Leu Phe Arg 290 295 300 Phe Lys His Leu Ala Thr Gly His Tyr Leu Ala Ala Glu Val Asp Pro 305 310 315 320 Asp Phe Glu Glu Glu Cys Leu Glu Phe Gln Pro Ser Val Asp Pro Asp 325 330 335 Gln Asp Ala Ser Arg Ser Arg Leu Arg Asn Ala Gln Glu Lys Met Val 340 345 350 Tyr Ser Leu Val Ser Val Pro Glu Gly Asn Asp Ile Ser Ser Ile Phe 355 360 365 Glu Leu Asp Pro Thr Thr Leu Arg Gly Gly Asp Ser Leu Val Pro Arg 370 375 380 Asn Ser Tyr Val Arg Leu Arg His Leu Cys Thr Asn Thr Trp Val His 385 390 395 400 Ser Thr Asn Ile Pro Ile Asp Lys Glu Glu Glu Lys Pro Val Met Leu 405 410 415 Lys Ile Gly Thr Ser Pro Leu Lys Glu Asp Lys Glu Ala Phe Ala Ile 420 425 430 Val Pro Val Ser Pro Ala Glu Val Arg Asp Leu Asp Phe Ala Asn Asp 435 440 445 Ala Ser Lys Val Leu Gly Ser Ile Ala Gly Lys Leu Glu Lys Gly Thr 450 455 460 Ile Thr Gln Asn Glu Arg Arg Ser Val Thr Lys Leu Leu Glu Asp Leu 465 470 475 480 Val Tyr Phe Val Thr Gly Gly Thr Asn Ser Gly Gln Asp Val Leu Glu 485 490 495 Val Val Phe Ser Lys Pro Asn Arg Glu Arg Gln Lys Leu Met Arg Glu 500 505 510 Gln Asn Ile Leu Lys Gln Ile Phe Lys Leu Leu Gln Ala Pro Phe Thr 515 520 525 Asp Cys Gly Asp Gly Pro Met Leu Arg Leu Glu Glu Leu Gly Asp Gln 530 535 540 Arg His Ala Pro Phe Arg His Ile Cys Arg Leu Cys Tyr Arg Val Leu 545 550 555 560 Arg His Ser Gln Gln Asp Tyr Arg Lys Asn Gln Glu Tyr Ile Ala Lys 565 570 575 Gln Phe Gly Phe Met Gln Lys Gln Ile Gly Tyr Asp Val Leu Ala Glu 580 585 590 Asp Thr Ile Thr Ala Leu Leu His Asn Asn Arg Lys 595 600 3 9848 DNA Mus musculus CDS (329)..(8575) 3 gctgaagcgt ttcctcaagc ctgccggggt gggaggagag gaggaggtgg tggtggtgga 60 ggaggtggag gcagagggtg gagagagaga aagcgcacgc cgagaggagg tgtgggtgtt 120 ccgctcccat cctaacggaa cgagctccct cttcgcggac atgggattgc ccagcggctg 180 ctaacccctc tcctggtcct gatcccccaa accggcgtgg ctccccggtc accaaggagc 240 tgattacaag ggaccaggat ttgcatcctt ggctgggcgt ccattggcta cagagtgcct 300 gacctgggtc aggctttcca acacggac atg tct gac aaa atg tcg agt ttc 352 Met Ser Asp Lys Met Ser Ser Phe 1 5 cta cat att gga gac att tgt tct ctg tat gcg gag gga tct acg aat 400 Leu His Ile Gly Asp Ile Cys Ser Leu Tyr Ala Glu Gly Ser Thr Asn 10 15 20 gga ttt atc agc acc tta ggc ttg gtt gat gac cgt tgt gtt gta cag 448 Gly Phe Ile Ser Thr Leu Gly Leu Val Asp Asp Arg Cys Val Val Gln 25 30 35 40 cca gaa gcc ggg gac ctt aac aat cca ccc aag aaa ttc aga gac tgc 496 Pro Glu Ala Gly Asp Leu Asn Asn Pro Pro Lys Lys Phe Arg Asp Cys 45 50 55 ctc ttt aag cta tgt cct atg aat cga tac tcc gca cag aaa cag ttc 544 Leu Phe Lys Leu Cys Pro Met Asn Arg Tyr Ser Ala Gln Lys Gln Phe 60 65 70 tgg aaa gct gct aag ccc ggg gcc aac agc act aca gat gca gtg ctg 592 Trp Lys Ala Ala Lys Pro Gly Ala Asn Ser Thr Thr Asp Ala Val Leu 75 80 85 ctc aac aaa ttg cat cat gct gca gac ttg gaa aag aag cag aat gag 640 Leu Asn Lys Leu His His Ala Ala Asp Leu Glu Lys Lys Gln Asn Glu 90 95 100 aca gaa aac agg aaa ttg ttg ggg acc gtc atc caa tat ggc aac gtg 688 Thr Glu Asn Arg Lys Leu Leu Gly Thr Val Ile Gln Tyr Gly Asn Val 105 110 115 120 atc cag ctc ctg cat ttg aaa agc aat aaa tac ctg act gtg aat aag 736 Ile Gln Leu Leu His Leu Lys Ser Asn Lys Tyr Leu Thr Val Asn Lys 125 130 135 agg ctc cca gcc ttg cta gag aag aat gcc atg agg gtg acg ttg gac 784 Arg Leu Pro Ala Leu Leu Glu Lys Asn Ala Met Arg Val Thr Leu Asp 140 145 150 gag gct gga aat gaa ggg tcc tgg ttt tac att caa cca ttt tac aag 832 Glu Ala Gly Asn Glu Gly Ser Trp Phe Tyr Ile Gln Pro Phe Tyr Lys 155 160 165 ctt cgc tcc atc gga gac agt gtg gtc ata ggc gac aag gta gtt ttg 880 Leu Arg Ser Ile Gly Asp Ser Val Val Ile Gly Asp Lys Val Val Leu 170 175 180 aat cct gtc aat gct ggc cag cct cta cat gcc agc agt cat cag ctg 928 Asn Pro Val Asn Ala Gly Gln Pro Leu His Ala Ser Ser His Gln Leu 185 190 195 200 gtg gat aac cca ggc tgc aat gag gtc aac tcc gtc aac tgt aat aca 976 Val Asp Asn Pro Gly Cys Asn Glu Val Asn Ser Val Asn Cys Asn Thr 205 210 215 agc tgg aag ata gtg ctt ttc atg aaa tgg agt gat aac aaa gac gac 1024 Ser Trp Lys Ile Val Leu Phe Met Lys Trp Ser Asp Asn Lys Asp Asp 220 225 230 att ctc aaa gga ggt gat gtg gtg agg ctc ttc cat gcc gag caa gag 1072 Ile Leu Lys Gly Gly Asp Val Val Arg Leu Phe His Ala Glu Gln Glu 235 240 245 aag ttt ctc acc tgt gat gag cac cgg aag aag cag cat gtg ttc ctg 1120 Lys Phe Leu Thr Cys Asp Glu His Arg Lys Lys Gln His Val Phe Leu 250 255 260 agg acc acc ggc agg cag tca gcc acg tcg gcc acc agt tct aaa gcc 1168 Arg Thr Thr Gly Arg Gln Ser Ala Thr Ser Ala Thr Ser Ser Lys Ala 265 270 275 280 ctg tgg gaa gtg gag gta gtc cag cac gac cca tgt cgg ggt gga gct 1216 Leu Trp Glu Val Glu Val Val Gln His Asp Pro Cys Arg Gly Gly Ala 285 290 295 ggg tac tgg aat agc ctc ttc cgg ttc aag cac ctg gct aca ggg cat 1264 Gly Tyr Trp Asn Ser Leu Phe Arg Phe Lys His Leu Ala Thr Gly His 300 305 310 tac ttg gct gca gag gta gac cct gac ttt gag gaa gaa tgc ctg gag 1312 Tyr Leu Ala Ala Glu Val Asp Pro Asp Phe Glu Glu Glu Cys Leu Glu 315 320 325 ttt cag ccc tca gtg gac cct gat cag gat gca tct cgg agt agg ttg 1360 Phe Gln Pro Ser Val Asp Pro Asp Gln Asp Ala Ser Arg Ser Arg Leu 330 335 340 aga aac gcg caa gaa aaa atg gta tac tct ctg gtc tcc gtg cct gaa 1408 Arg Asn Ala Gln Glu Lys Met Val Tyr Ser Leu Val Ser Val Pro Glu 345 350 355 360 ggc aac gac atc tcc tcc atc ttt gag cta gac ccc acg act ctg cgt 1456 Gly Asn Asp Ile Ser Ser Ile Phe Glu Leu Asp Pro Thr Thr Leu Arg 365 370 375 gga ggt gac agc ctt gtc cca agg aac tcc tat gtc cgt ctc aga cac 1504 Gly Gly Asp Ser Leu Val Pro Arg Asn Ser Tyr Val Arg Leu Arg His 380 385 390 ctg tgc acc aac acc tgg gta cac agc aca aac atc ccc atc gac aag 1552 Leu Cys Thr Asn Thr Trp Val His Ser Thr Asn Ile Pro Ile Asp Lys 395 400 405 gaa gag gag aag cct gtg atg ctg aaa att ggt acc tct ccc ctg aag 1600 Glu Glu Glu Lys Pro Val Met Leu Lys Ile Gly Thr Ser Pro Leu Lys 410 415 420 gag gac aag gaa gca ttt gcc ata gtt cct gtt tcc cct gct gag gtt 1648 Glu Asp Lys Glu Ala Phe Ala Ile Val Pro Val Ser Pro Ala Glu Val 425 430 435 440 cgg gac ctg gac ttt gcc aat gat gcc agc aag gtg ctg ggc tcc atc 1696 Arg Asp Leu Asp Phe Ala Asn Asp Ala Ser Lys Val Leu Gly Ser Ile 445 450 455 gct ggg aag ttg gaa aag ggc acc atc acc cag aat gag aga agg tct 1744 Ala Gly Lys Leu Glu Lys Gly Thr Ile Thr Gln Asn Glu Arg Arg Ser 460 465 470 gtc acg aag ctt ttg gaa gac ttg gtt tac ttt gtc acg ggt gga act 1792 Val Thr Lys Leu Leu Glu Asp Leu Val Tyr Phe Val Thr Gly Gly Thr 475 480 485 aac tct ggc caa gac gtg ctt gaa gtt gtc ttc tct aag ccc aat cga 1840 Asn Ser Gly Gln Asp Val Leu Glu Val Val Phe Ser Lys Pro Asn Arg 490 495 500 gag cgg cag aag ctg atg agg gaa cag aat att ctc aag cag atc ttc 1888 Glu Arg Gln Lys Leu Met Arg Glu Gln Asn Ile Leu Lys Gln Ile Phe 505 510 515 520 aag ctg ttg cag gcc ccc ttc acg gac tgc ggg gat ggc ccg atg ctt 1936 Lys Leu Leu Gln Ala Pro Phe Thr Asp Cys Gly Asp Gly Pro Met Leu 525 530 535 cgg ctg gag gag ctg ggg gat cag cgc cat gct cct ttc aga cat att 1984 Arg Leu Glu Glu Leu Gly Asp Gln Arg His Ala Pro Phe Arg His Ile 540 545 550 tgc cga ctc tgc tac agg gtc ctg cga cac tca cag caa gac tac agg 2032 Cys Arg Leu Cys Tyr Arg Val Leu Arg His Ser Gln Gln Asp Tyr Arg 555 560 565 aag aac cag gag tac ata gcc aag cag ttt ggc ttc atg cag aag cag 2080 Lys Asn Gln Glu Tyr Ile Ala Lys Gln Phe Gly Phe Met Gln Lys Gln 570 575 580 att ggc tat gac gtg ctg gcc gaa gac acc atc act gcc ctg ctc cac 2128 Ile Gly Tyr Asp Val Leu Ala Glu Asp Thr Ile Thr Ala Leu Leu His 585 590 595 600 aac aac cgg aaa ctc ctg gag aag cac atc acc gcg gca gag att gac 2176 Asn Asn Arg Lys Leu Leu Glu Lys His Ile Thr Ala Ala Glu Ile Asp 605 610 615 acg ttt gtc agc ctg gtg cga aag aac agg gag ccc agg ttc ttg gat 2224 Thr Phe Val Ser Leu Val Arg Lys Asn Arg Glu Pro Arg Phe Leu Asp 620 625 630 tac ctc tct gac ctc tgc gta tcc atg aac aag tca atc cct gtg aca 2272 Tyr Leu Ser Asp Leu Cys Val Ser Met Asn Lys Ser Ile Pro Val Thr 635 640 645 cag gag ctc atc tgt aaa gct gtg ctc aat ccc acc aat gct gac atc 2320 Gln Glu Leu Ile Cys Lys Ala Val Leu Asn Pro Thr Asn Ala Asp Ile 650 655 660 ctg att gag acc aag ctg gtt ctt tct cgt ttt gag ttt gaa ggc gtt 2368 Leu Ile Glu Thr Lys Leu Val Leu Ser Arg Phe Glu Phe Glu Gly Val 665 670 675 680 tcc act gga gag aat gct ctg gaa gcc ggg gag gat gag gaa gag gtg 2416 Ser Thr Gly Glu Asn Ala Leu Glu Ala Gly Glu Asp Glu Glu Glu Val 685 690 695 tgg ctg ttc tgg agg gac agc aac aaa gag atc cgt agt aag agt gtc 2464 Trp Leu Phe Trp Arg Asp Ser Asn Lys Glu Ile Arg Ser Lys Ser Val 700 705 710 cgg gaa ttg gcg caa gat gct aaa gag gga cag aag gaa gac agg gac 2512 Arg Glu Leu Ala Gln Asp Ala Lys Glu Gly Gln Lys Glu Asp Arg Asp 715 720 725 atc ctc agc tac tac aga tat cag ctg aac ctc ttt gca agg atg tgt 2560 Ile Leu Ser Tyr Tyr Arg Tyr Gln Leu Asn Leu Phe Ala Arg Met Cys 730 735 740 ctg gac cgc cag tac ctg gcc atc aat gaa atc tcc ggg cag ctg gat 2608 Leu Asp Arg Gln Tyr Leu Ala Ile Asn Glu Ile Ser Gly Gln Leu Asp 745 750 755 760 gtt gat ctc att ctc cgc tgc atg tct gac gag aac ctc ccc tac gac 2656 Val Asp Leu Ile Leu Arg Cys Met Ser Asp Glu Asn Leu Pro Tyr Asp 765 770 775 ctc agg gca tcc ttt tgc cgc ctc atg ctt cac atg cat gtg gac cga 2704 Leu Arg Ala Ser Phe Cys Arg Leu Met Leu His Met His Val Asp Arg 780 785 790 gat ccc caa gag cag gtg aca cct gtg aaa tat gcc cga ctg tgg tca 2752 Asp Pro Gln Glu Gln Val Thr Pro Val Lys Tyr Ala Arg Leu Trp Ser 795 800 805 gaa att ccc tct gag atc gcc att gat gac tat gac agc agt gga aca 2800 Glu Ile Pro Ser Glu Ile Ala Ile Asp Asp Tyr Asp Ser Ser Gly Thr 810 815 820 tcc aaa gat gaa att aag gag agg ttt gca cag acg atg gag ttt gtg 2848 Ser Lys Asp Glu Ile Lys Glu Arg Phe Ala Gln Thr Met Glu Phe Val 825 830 835 840 gag gag tac cta aga gat gtg gtt tgt caa aga ttc ccc ttc tct gat 2896 Glu Glu Tyr Leu Arg Asp Val Val Cys Gln Arg Phe Pro Phe Ser Asp 845 850 855 aag gag aaa aat aag ctc acg ttt gag gtt gtg aac tta gcc agg aat 2944 Lys Glu Lys Asn Lys Leu Thr Phe Glu Val Val Asn Leu Ala Arg Asn 860 865 870 ctc ata tac ttt ggt ttc tac aac ttt tct gac ctt ctc cga tta acc 2992 Leu Ile Tyr Phe Gly Phe Tyr Asn Phe Ser Asp Leu Leu Arg Leu Thr 875 880 885 aag atc ctc ttg gca atc tta gac tgt gtc cat gtg acc act atc ttc 3040 Lys Ile Leu Leu Ala Ile Leu Asp Cys Val His Val Thr Thr Ile Phe 890 895 900 ccc att agc aag atg aca aaa gga gaa gag aat aaa ggc agt aac gtg 3088 Pro Ile Ser Lys Met Thr Lys Gly Glu Glu Asn Lys Gly Ser Asn Val 905 910 915 920 atg agg tct atc cat ggc gtt ggg gag ctg atg acc cag gtg gtg ctg 3136 Met Arg Ser Ile His Gly Val Gly Glu Leu Met Thr Gln Val Val Leu 925 930 935 cgg gga gga ggc ttc ttg ccc atg act ccc atg gct gcg gcc cct gaa 3184 Arg Gly Gly Gly Phe Leu Pro Met Thr Pro Met Ala Ala Ala Pro Glu 940 945 950 gga aat gtg aag cag gca gag cca gag aaa gag gac atc atg gtc atg 3232 Gly Asn Val Lys Gln Ala Glu Pro Glu Lys Glu Asp Ile Met Val Met 955 960 965 gac acc aag ttg aag atc att gaa ata ctc cag ttt att ttg aat gtg 3280 Asp Thr Lys Leu Lys Ile Ile Glu Ile Leu Gln Phe Ile Leu Asn Val 970 975 980 aga ttg gat tat agg atc tcc tgc ctc ctg tgt ata ttt aag cga gag 3328 Arg Leu Asp Tyr Arg Ile Ser Cys Leu Leu Cys Ile Phe Lys Arg Glu 985 990 995 1000 ttt gat gaa agc aat tcc cag tca tca gaa aca tcc tcc gga aac agc 3376 Phe Asp Glu Ser Asn Ser Gln Ser Ser Glu Thr Ser Ser Gly Asn Ser 1005 1010 1015 agc cag gaa ggg cca agt aat gtg cca ggt gct ctt gac ttt gaa cac 3424 Ser Gln Glu Gly Pro Ser Asn Val Pro Gly Ala Leu Asp Phe Glu His 1020 1025 1030 att gaa gaa caa gcg gaa ggc atc ttt gga gga agt gag gag aac aca 3472 Ile Glu Glu Gln Ala Glu Gly Ile Phe Gly Gly Ser Glu Glu Asn Thr 1035 1040 1045 cct ttg gac ctg gat gac cat ggt ggc aga acc ttc ctc agg gtc ctg 3520 Pro Leu Asp Leu Asp Asp His Gly Gly Arg Thr Phe Leu Arg Val Leu 1050 1055 1060 ctc cac ttg aca atg cat gac tac cca ccc ctg gtg tct ggg gcc ctg 3568 Leu His Leu Thr Met His Asp Tyr Pro Pro Leu Val Ser Gly Ala Leu 1065 1070 1075 1080 cag ctc ctc ttt cgg cac ttc agc cag agg cag gag gtc ctt cag gcc 3616 Gln Leu Leu Phe Arg His Phe Ser Gln Arg Gln Glu Val Leu Gln Ala 1085 1090 1095 ttc aaa cag gtt caa ctg ctg gtt act agc caa gat gtg gac aac tac 3664 Phe Lys Gln Val Gln Leu Leu Val Thr Ser Gln Asp Val Asp Asn Tyr 1100 1105 1110 aaa cag atc aag caa gac ttg gac caa cta agg tcc att gtg gag aag 3712 Lys Gln Ile Lys Gln Asp Leu Asp Gln Leu Arg Ser Ile Val Glu Lys 1115 1120 1125 tct gag ctc tgg gtg tac aaa ggc caa ggt ccc gat gag cct atg gac 3760 Ser Glu Leu Trp Val Tyr Lys Gly Gln Gly Pro Asp Glu Pro Met Asp 1130 1135 1140 gga gcc tcc ggt gaa aat gag cat aag aaa acc gag gag ggg acg agc 3808 Gly Ala Ser Gly Glu Asn Glu His Lys Lys Thr Glu Glu Gly Thr Ser 1145 1150 1155 1160 aag cca ctg aag cac gag agc acc agc agc tac aac tac cga gtg gtg 3856 Lys Pro Leu Lys His Glu Ser Thr Ser Ser Tyr Asn Tyr Arg Val Val 1165 1170 1175 aaa gag att ttg att cga ctt agc aag ctc tgc gtg cag gag agc gcg 3904 Lys Glu Ile Leu Ile Arg Leu Ser Lys Leu Cys Val Gln Glu Ser Ala 1180 1185 1190 tcg gtg agg aag agc cgg aag cag cag caa cga ctg ctg agg aac atg 3952 Ser Val Arg Lys Ser Arg Lys Gln Gln Gln Arg Leu Leu Arg Asn Met 1195 1200 1205 ggc gca cac gct gtg gtg ctg gag ctg ctg cag atc ccc tac gag aag 4000 Gly Ala His Ala Val Val Leu Glu Leu Leu Gln Ile Pro Tyr Glu Lys 1210 1215 1220 gcc gaa gac aca aag atg caa gag atc atg cgg ctg gct cat gaa ttt 4048 Ala Glu Asp Thr Lys Met Gln Glu Ile Met Arg Leu Ala His Glu Phe 1225 1230 1235 1240 ttg cag aat ttc tgt gca ggc aac cag cag aat caa gct ttg ctg cat 4096 Leu Gln Asn Phe Cys Ala Gly Asn Gln Gln Asn Gln Ala Leu Leu His 1245 1250 1255 aaa cac ata aac ctg ttt ctc aag cca ggg atc ctg gag gca gtg acg 4144 Lys His Ile Asn Leu Phe Leu Lys Pro Gly Ile Leu Glu Ala Val Thr 1260 1265 1270 atg cag cac atc ttc atg aac aac ttc cag ctg tgc agt gag atc aac 4192 Met Gln His Ile Phe Met Asn Asn Phe Gln Leu Cys Ser Glu Ile Asn 1275 1280 1285 gag aga gtg gtc cag cac ttt gtt cac tgc ata gag acc cac ggt cga 4240 Glu Arg Val Val Gln His Phe Val His Cys Ile Glu Thr His Gly Arg 1290 1295 1300 aac gtc cag tat atc aag ttt ctc cag acg att gtc aag gca gaa ggg 4288 Asn Val Gln Tyr Ile Lys Phe Leu Gln Thr Ile Val Lys Ala Glu Gly 1305 1310 1315 1320 aaa ttc att aaa aag tgc caa gac atg gtc atg gct gag ctt gtc aac 4336 Lys Phe Ile Lys Lys Cys Gln Asp Met Val Met Ala Glu Leu Val Asn 1325 1330 1335 tct gga gag gac gtc ctc gtg ttc tac aat gac aga gcc tct ttc cag 4384 Ser Gly Glu Asp Val Leu Val Phe Tyr Asn Asp Arg Ala Ser Phe Gln 1340 1345 1350 act ctg atc cag atg atg cgg tcc gag cgt gac cgg atg gat gag aac 4432 Thr Leu Ile Gln Met Met Arg Ser Glu Arg Asp Arg Met Asp Glu Asn 1355 1360 1365 agc cct ctc atg tac cac atc cat ctg gtg gag ctc ttg gcc gtg tgc 4480 Ser Pro Leu Met Tyr His Ile His Leu Val Glu Leu Leu Ala Val Cys 1370 1375 1380 aca gag ggc aag aat gtg tac acg gag atc aag tgc aac tcc ttg ctc 4528 Thr Glu Gly Lys Asn Val Tyr Thr Glu Ile Lys Cys Asn Ser Leu Leu 1385 1390 1395 1400 ccg ctc gat gac atc gtt cgt gtg gtc act cat gaa gac tgc atc ccc 4576 Pro Leu Asp Asp Ile Val Arg Val Val Thr His Glu Asp Cys Ile Pro 1405 1410 1415 gag gtt aag atc gct tac att aac ttc ctg aat cac tgc tat gtg gat 4624 Glu Val Lys Ile Ala Tyr Ile Asn Phe Leu Asn His Cys Tyr Val Asp 1420 1425 1430 acg gag gtg gag atg aag gag att tac aca agc aac cac atg tgg aag 4672 Thr Glu Val Glu Met Lys Glu Ile Tyr Thr Ser Asn His Met Trp Lys 1435 1440 1445 ttg ttt gag aat ttc ctc gtg gac atc tgc agg gcc tgt aac aac aca 4720 Leu Phe Glu Asn Phe Leu Val Asp Ile Cys Arg Ala Cys Asn Asn Thr 1450 1455 1460 agc gac agg aag cac gca gac tcc att ctg gag aag tac gtc act gaa 4768 Ser Asp Arg Lys His Ala Asp Ser Ile Leu Glu Lys Tyr Val Thr Glu 1465 1470 1475 1480 atc gtg atg agc atc gtc acc acc ttc ttc agc tct ccc ttc tca gac 4816 Ile Val Met Ser Ile Val Thr Thr Phe Phe Ser Ser Pro Phe Ser Asp 1485 1490 1495 cag agc acc act ctg cag acc cgc cag cct gtc ttt gtg caa ctc ctg 4864 Gln Ser Thr Thr Leu Gln Thr Arg Gln Pro Val Phe Val Gln Leu Leu 1500 1505 1510 caa ggc gtg ttc cga gtt tac cac tgc aac tgg ctg atg ccg agc caa 4912 Gln Gly Val Phe Arg Val Tyr His Cys Asn Trp Leu Met Pro Ser Gln 1515 1520 1525 aaa gcc tcg gtg gag agc tgc atc cgg gtg ctc tct gac gta gcc aag 4960 Lys Ala Ser Val Glu Ser Cys Ile Arg Val Leu Ser Asp Val Ala Lys 1530 1535 1540 agc cgg gcc ata gcc att cct gtt gac ctg gac agc caa gtc aac aac 5008 Ser Arg Ala Ile Ala Ile Pro Val Asp Leu Asp Ser Gln Val Asn Asn 1545 1550 1555 1560 ctc ttc ctg aag tcc cac aac att gtg cag aaa aca gcc ctg aac tgg 5056 Leu Phe Leu Lys Ser His Asn Ile Val Gln Lys Thr Ala Leu Asn Trp 1565 1570 1575 cgg tta tca gcc cga aac gcc gct cgc aga gac tct gta ctg gca gca 5104 Arg Leu Ser Ala Arg Asn Ala Ala Arg Arg Asp Ser Val Leu Ala Ala 1580 1585 1590 tcc aga gac tac cga aat atc att gag agg tta cag gac atc gtg tct 5152 Ser Arg Asp Tyr Arg Asn Ile Ile Glu Arg Leu Gln Asp Ile Val Ser 1595 1600 1605 gcc cta gag gac cgg ctc agg ccc ctg gtg cag gct gag ctg tct gtg 5200 Ala Leu Glu Asp Arg Leu Arg Pro Leu Val Gln Ala Glu Leu Ser Val 1610 1615 1620 ctc gtg gat gtt cta cac aga cca gaa ctg ctc ttc ccc gag aac acg 5248 Leu Val Asp Val Leu His Arg Pro Glu Leu Leu Phe Pro Glu Asn Thr 1625 1630 1635 1640 gat gcc agg agg aaa tgt gag agt gga ggt ttc atc tgc aag cta ata 5296 Asp Ala Arg Arg Lys Cys Glu Ser Gly Gly Phe Ile Cys Lys Leu Ile 1645 1650 1655 aaa cat acc aag caa ctg ctg gag gag aat gaa gag aaa cta tgc att 5344 Lys His Thr Lys Gln Leu Leu Glu Glu Asn Glu Glu Lys Leu Cys Ile 1660 1665 1670 aaa gtc tta cag acc ctc agg gaa atg atg acc aaa gac aga ggc tat 5392 Lys Val Leu Gln Thr Leu Arg Glu Met Met Thr Lys Asp Arg Gly Tyr 1675 1680 1685 gga gag aag caa att tcc att gat gaa tcg gaa aat gcc gag ctg cca 5440 Gly Glu Lys Gln Ile Ser Ile Asp Glu Ser Glu Asn Ala Glu Leu Pro 1690 1695 1700 cag gca ccg gaa gct gag aac tcc aca gag cag gag ctt gaa cca agt 5488 Gln Ala Pro Glu Ala Glu Asn Ser Thr Glu Gln Glu Leu Glu Pro Ser 1705 1710 1715 1720 cca ccc ctg agg caa ctg gaa gac cat aaa agg ggt gag gca ctc cga 5536 Pro Pro Leu Arg Gln Leu Glu Asp His Lys Arg Gly Glu Ala Leu Arg 1725 1730 1735 caa att ttg gtc aac cgt tac tat gga aac atc aga cct tca gga aga 5584 Gln Ile Leu Val Asn Arg Tyr Tyr Gly Asn Ile Arg Pro Ser Gly Arg 1740 1745 1750 aga gag agc ctt acc agc ttt ggc aat ggc cca cta tca cca gga gga 5632 Arg Glu Ser Leu Thr Ser Phe Gly Asn Gly Pro Leu Ser Pro Gly Gly 1755 1760 1765 ccc agc aag cct ggt gga gga ggg gga ggt cct gga tct agt tcc aca 5680 Pro Ser Lys Pro Gly Gly Gly Gly Gly Gly Pro Gly Ser Ser Ser Thr 1770 1775 1780 agc agg ggt gag atg agc ctg gct gag gtt cag tgt cac ctc gac aag 5728 Ser Arg Gly Glu Met Ser Leu Ala Glu Val Gln Cys His Leu Asp Lys 1785 1790 1795 1800 gag ggg gcc tcc aac ctg gtc atc gat ctc ata atg aat gca tcc agt 5776 Glu Gly Ala Ser Asn Leu Val Ile Asp Leu Ile Met Asn Ala Ser Ser 1805 1810 1815 gac cga gta ttc cat gaa agc att ctg ctg gcc atc gca ctt ctg gaa 5824 Asp Arg Val Phe His Glu Ser Ile Leu Leu Ala Ile Ala Leu Leu Glu 1820 1825 1830 gga ggc aac acc acc atc cag cac tcg ttt ttc tgc cgg ctg aca gaa 5872 Gly Gly Asn Thr Thr Ile Gln His Ser Phe Phe Cys Arg Leu Thr Glu 1835 1840 1845 gat aag aaa tca gag aag ttc ttc aag gtt ttt tac gat cga atg aag 5920 Asp Lys Lys Ser Glu Lys Phe Phe Lys Val Phe Tyr Asp Arg Met Lys 1850 1855 1860 gtg gcc cag cag gaa atc aag gcg aca gtg aca gtg aac acc agc gac 5968 Val Ala Gln Gln Glu Ile Lys Ala Thr Val Thr Val Asn Thr Ser Asp 1865 1870 1875 1880 ttg gga aac aaa aag aaa gat gat gaa gtg gac agg gat gcc ccg tct 6016 Leu Gly Asn Lys Lys Lys Asp Asp Glu Val Asp Arg Asp Ala Pro Ser 1885 1890 1895 cgg aag aaa gcc aaa gag ccc aca aca cag ata aca gaa gag gtc cgg 6064 Arg Lys Lys Ala Lys Glu Pro Thr Thr Gln Ile Thr Glu Glu Val Arg 1900 1905 1910 gat cag ctc ctg gaa gca tct gct gcc acc agg aaa gcc ttt acc acc 6112 Asp Gln Leu Leu Glu Ala Ser Ala Ala Thr Arg Lys Ala Phe Thr Thr 1915 1920 1925 ttc cgg agg gag gcc gac cct gat gac cat tac cag tct ggg gag ggc 6160 Phe Arg Arg Glu Ala Asp Pro Asp Asp His Tyr Gln Ser Gly Glu Gly 1930 1935 1940 acc cag gct aca acc gac aaa gcc aag gat gac cta gag atg agc gct 6208 Thr Gln Ala Thr Thr Asp Lys Ala Lys Asp Asp Leu Glu Met Ser Ala 1945 1950 1955 1960 gtc atc acc atc atg cag cct atc ctg cgc ttc ctg cag ctg ctg tgt 6256 Val Ile Thr Ile Met Gln Pro Ile Leu Arg Phe Leu Gln Leu Leu Cys 1965 1970 1975 gaa aac cac aac cga gat ctg cag aat ttc ctt cgt tgc caa aat aat 6304 Glu Asn His Asn Arg Asp Leu Gln Asn Phe Leu Arg Cys Gln Asn Asn 1980 1985 1990 aag acc aac tac aat ttg gtg tgt gag aca ctg cag ttt ctg gac tgt 6352 Lys Thr Asn Tyr Asn Leu Val Cys Glu Thr Leu Gln Phe Leu Asp Cys 1995 2000 2005 att tgt ggg agc aca acc gga ggc ctt ggt ctt ctt gga ctg tac ata 6400 Ile Cys Gly Ser Thr Thr Gly Gly Leu Gly Leu Leu Gly Leu Tyr Ile 2010 2015 2020 aat gaa aag aat gta gca ctt atc aac caa acc ctg gag agt ctg acg 6448 Asn Glu Lys Asn Val Ala Leu Ile Asn Gln Thr Leu Glu Ser Leu Thr 2025 2030 2035 2040 gag tac tgt caa ggg cct tgc cat gag aac cag aac tgc atc gcc acc 6496 Glu Tyr Cys Gln Gly Pro Cys His Glu Asn Gln Asn Cys Ile Ala Thr 2045 2050 2055 cac gag tcc aat ggc atc gat atc atc aca gcc ctc atc ctc aat gat 6544 His Glu Ser Asn Gly Ile Asp Ile Ile Thr Ala Leu Ile Leu Asn Asp 2060 2065 2070 atc aac cct ctg gga aag aag cgg atg gac ctg gtg tta gaa ctg aag 6592 Ile Asn Pro Leu Gly Lys Lys Arg Met Asp Leu Val Leu Glu Leu Lys 2075 2080 2085 aac aat gct tcg aag ctg cta ctg gcc atc atg gaa agc aga cac gat 6640 Asn Asn Ala Ser Lys Leu Leu Leu Ala Ile Met Glu Ser Arg His Asp 2090 2095 2100 agt gaa aat gca gag agg atc ctg tac aac atg agg ccc aag gag ctg 6688 Ser Glu Asn Ala Glu Arg Ile Leu Tyr Asn Met Arg Pro Lys Glu Leu 2105 2110 2115 2120 gtg gaa gtg atc aag aag gcc tac atg caa ggt gaa gtg gaa ttt gag 6736 Val Glu Val Ile Lys Lys Ala Tyr Met Gln Gly Glu Val Glu Phe Glu 2125 2130 2135 gat ggg gag aac ggt gag gat gga gct gcc tca ccc agg aac gtg ggc 6784 Asp Gly Glu Asn Gly Glu Asp Gly Ala Ala Ser Pro Arg Asn Val Gly 2140 2145 2150 cac aac atc tac atc ctc gct cac cag ttg gct cgg cat aac aaa gaa 6832 His Asn Ile Tyr Ile Leu Ala His Gln Leu Ala Arg His Asn Lys Glu 2155 2160 2165 ctt caa acc atg ctg aaa cct gga ggc cag gtg gat ggg gat gaa gct 6880 Leu Gln Thr Met Leu Lys Pro Gly Gly Gln Val Asp Gly Asp Glu Ala 2170 2175 2180 ctg gag ttc tac gcg aag cac aca gca caa att gag att gtc aga ctg 6928 Leu Glu Phe Tyr Ala Lys His Thr Ala Gln Ile Glu Ile Val Arg Leu 2185 2190 2195 2200 gac cgg aca atg gaa cag atc gtc ttc cct gtg ccc agc atc tgt gaa 6976 Asp Arg Thr Met Glu Gln Ile Val Phe Pro Val Pro Ser Ile Cys Glu 2205 2210 2215 ttc ctg act aag gaa tcg aaa ctt cga ata tat tac acc aca gag cgg 7024 Phe Leu Thr Lys Glu Ser Lys Leu Arg Ile Tyr Tyr Thr Thr Glu Arg 2220 2225 2230 gat gag caa ggt agc aag atc aat gac ttc ttc ctg cgc tcc gag gac 7072 Asp Glu Gln Gly Ser Lys Ile Asn Asp Phe Phe Leu Arg Ser Glu Asp 2235 2240 2245 ctc ttt aac gag atg aac tgg cag aag aaa ctt cga gcc cag cct gtc 7120 Leu Phe Asn Glu Met Asn Trp Gln Lys Lys Leu Arg Ala Gln Pro Val 2250 2255 2260 ttg tac tgg tgt gcc cga aac atg tct ttc tgg agc agc atc tcc ttc 7168 Leu Tyr Trp Cys Ala Arg Asn Met Ser Phe Trp Ser Ser Ile Ser Phe 2265 2270 2275 2280 aac ctg gcc gtc ctg atg aac ctg ctg gtg gcg ttt ttc tat cca ttt 7216 Asn Leu Ala Val Leu Met Asn Leu Leu Val Ala Phe Phe Tyr Pro Phe 2285 2290 2295 aaa gga gtg agg gga gga aca cta gag cca cac tgg tca ggc ctc ctg 7264 Lys Gly Val Arg Gly Gly Thr Leu Glu Pro His Trp Ser Gly Leu Leu 2300 2305 2310 tgg aca gcc atg ctc atc tct ctg gcc att gtc att gct ctg ccc aag 7312 Trp Thr Ala Met Leu Ile Ser Leu Ala Ile Val Ile Ala Leu Pro Lys 2315 2320 2325 ccc cac ggc atc cgg gcc tta att gct tct aca atc cta cga ctg ata 7360 Pro His Gly Ile Arg Ala Leu Ile Ala Ser Thr Ile Leu Arg Leu Ile 2330 2335 2340 ttt tca gtt ggg ttg cag ccc aca ctg ttt ctg ctg gga gct ttc aat 7408 Phe Ser Val Gly Leu Gln Pro Thr Leu Phe Leu Leu Gly Ala Phe Asn 2345 2350 2355 2360 gtc tgc aat aaa atc atc ttc ctg atg agc ttt gtg ggc aac tgt ggg 7456 Val Cys Asn Lys Ile Ile Phe Leu Met Ser Phe Val Gly Asn Cys Gly 2365 2370 2375 acc ttc acc aga ggc tac cgg gcc atg gtt ctg gat gtg gag ttc ctc 7504 Thr Phe Thr Arg Gly Tyr Arg Ala Met Val Leu Asp Val Glu Phe Leu 2380 2385 2390 tat cat ttg ctg tat cta ctc atc tgt gcc atg ggc ctc ttc gta cat 7552 Tyr His Leu Leu Tyr Leu Leu Ile Cys Ala Met Gly Leu Phe Val His 2395 2400 2405 gag ttc ttc tat agc ttg ctg ctt ttt gat tta gtg tac aga gag gag 7600 Glu Phe Phe Tyr Ser Leu Leu Leu Phe Asp Leu Val Tyr Arg Glu Glu 2410 2415 2420 act ttg ctt aat gtc att aaa agt gtc acc cgc aat gga cgg tcc atc 7648 Thr Leu Leu Asn Val Ile Lys Ser Val Thr Arg Asn Gly Arg Ser Ile 2425 2430 2435 2440 atc ttg aca gcg gtc ctg gct ctg atc ctg gtt tac ctg ttc tca att 7696 Ile Leu Thr Ala Val Leu Ala Leu Ile Leu Val Tyr Leu Phe Ser Ile 2445 2450 2455 gtg ggc tat ctg ttc ttc aag gat gac ttt atc ttg gaa gta gat agg 7744 Val Gly Tyr Leu Phe Phe Lys Asp Asp Phe Ile Leu Glu Val Asp Arg 2460 2465 2470 ttg ccc aat gaa aca gct gtt cca gaa act ggc gag agt ttg gcc aac 7792 Leu Pro Asn Glu Thr Ala Val Pro Glu Thr Gly Glu Ser Leu Ala Asn 2475 2480 2485 gat ttc ctg tac tct gat gtg tgc agg gta gag acg ggg gag aac tgc 7840 Asp Phe Leu Tyr Ser Asp Val Cys Arg Val Glu Thr Gly Glu Asn Cys 2490 2495 2500 acc tct cct gca ccc aaa gaa gag ctg ctc cct gcc gaa gaa acg gaa 7888 Thr Ser Pro Ala Pro Lys Glu Glu Leu Leu Pro Ala Glu Glu Thr Glu 2505 2510 2515 2520 cag gat aag gaa cac acg tgt gag acc ctg ctc atg tgc atc gtc act 7936 Gln Asp Lys Glu His Thr Cys Glu Thr Leu Leu Met Cys Ile Val Thr 2525 2530 2535 gtt ctg agt cac ggg ctg cgg agt ggg gga ggg gta gga gac gtg ctc 7984 Val Leu Ser His Gly Leu Arg Ser Gly Gly Gly Val Gly Asp Val Leu 2540 2545 2550 agg aag cca tcc aaa gag gag cct ctg ttt gct gca agg gtg atc tac 8032 Arg Lys Pro Ser Lys Glu Glu Pro Leu Phe Ala Ala Arg Val Ile Tyr 2555 2560 2565 gac ctc ctc ttc ttc ttc atg gtc atc atc atc gtc ctg aac ctg att 8080 Asp Leu Leu Phe Phe Phe Met Val Ile Ile Ile Val Leu Asn Leu Ile 2570 2575 2580 ttc ggg gtc atc atc gac acc ttt gct gac ctg agg agt gag aag caa 8128 Phe Gly Val Ile Ile Asp Thr Phe Ala Asp Leu Arg Ser Glu Lys Gln 2585 2590 2595 2600 aag aag gag gag atc tta aaa acc acg tgc ttc atc tgc ggc ttg gaa 8176 Lys Lys Glu Glu Ile Leu Lys Thr Thr Cys Phe Ile Cys Gly Leu Glu 2605 2610 2615 agg gac aag ttt gac aat aag act gtc acc ttt gaa gag cac atc aag 8224 Arg Asp Lys Phe Asp Asn Lys Thr Val Thr Phe Glu Glu His Ile Lys 2620 2625 2630 gaa gaa cac aac atg tgg cac tat ctg tgc ttc atc gtg ctg gtg aaa 8272 Glu Glu His Asn Met Trp His Tyr Leu Cys Phe Ile Val Leu Val Lys 2635 2640 2645 gtg aag gac tcc aca gag tac acc ggg cct gag agt tac gtg gca gag 8320 Val Lys Asp Ser Thr Glu Tyr Thr Gly Pro Glu Ser Tyr Val Ala Glu 2650 2655 2660 atg atc agg gaa aga aac ctt gat tgg ttc ctc aga atg aga gcc atg 8368 Met Ile Arg Glu Arg Asn Leu Asp Trp Phe Leu Arg Met Arg Ala Met 2665 2670 2675 2680 tcc ctg gtc agc agc gat tct gaa ggg gaa cag aac gag ctg agg aac 8416 Ser Leu Val Ser Ser Asp Ser Glu Gly Glu Gln Asn Glu Leu Arg Asn 2685 2690 2695 ctg cag gag aag ctg gag tct acc atg aag ctg gtc acc aat ctt tct 8464 Leu Gln Glu Lys Leu Glu Ser Thr Met Lys Leu Val Thr Asn Leu Ser 2700 2705 2710 ggc cag ctg tca gaa cta aag gac cag atg aca gaa cag agg aag cag 8512 Gly Gln Leu Ser Glu Leu Lys Asp Gln Met Thr Glu Gln Arg Lys Gln 2715 2720 2725 aaa caa aga atc ggc ctt cta gga cat cct cct cac atg aat gtc aac 8560 Lys Gln Arg Ile Gly Leu Leu Gly His Pro Pro His Met Asn Val Asn 2730 2735 2740 cca cag cag ccg gcc taggcaaatg aggcagaggg actctgctca gccctctgta 8615 Pro Gln Gln Pro Ala 2745 tatcactgtc agggtgggta cggctcattg gttctgattt gcccactaag ggtacatgtg 8675 cgcttagtac atttgtaaat actcagtttt gtattgtatg tatatgattg ctattctcag 8735 aggtttggac tttcgtattg taattagctc tgttggcatg gtgacttgtc actcctgcca 8795 aaaatattaa aaatgccttt tttggaagga ctacagaaag tacctgattt gcacttgaac 8855 cagattatag atttaaaagt atatgacatg tattttgtat ttaaaactag aatagccagt 8915 atttatgttt tttataaaac tgtgcaatac aaattatgca atcaccataa ctttgtaact 8975 cctgagtgtc ctaagggagt acacatcttt gaagctgatt tgttgatact cgtgtaataa 9035 atggttaaat atcaaatgct gctgctgctg ccaaaattat attaatagcg agtttctggc 9095 ccctgggcaa ttttgtacct tgtaattatc ctatggtgat gctgtttctc gttgctaatg 9155 gcattagtgc ccctgtatcc tagtgataac tccaggtctg tgaaccattc aaacagcatt 9215 cattttgaga aaagcaactt tagtttcaag gataatttta agcttcaaaa ttaatcattt 9275 aaagtgtttc tttaagagag ccatgttaga ggctcacact ttagcttgaa aggagttgat 9335 gaattaattt tttaaaggga actttttaca tgacgtttgg aataacagca tattgctgac 9395 cagtcagtgt catctcccgg gtgaattttg atgtcacgtt atagtcaaat gagttagctg 9455 atggtttcta gattttcttc ctctgaacca tgatgcagta ggtaagaagt tattatgcgt 9515 atatacatat atacattcat atacgacaaa gtaggagctg tccccttagg atgcatagct 9575 gcccctaggg tacgtagctg aacactgaca atggcgttct tctgaaagag ccacgtttgg 9635 gttttatttc tttgtcacat gatttctttt ctggatgggt gcaaagtatc acaggaagtg 9695 ttttctctct gtcgccttgt tttgtacctg ggtctcgctt tactagaccg tctctgcaca 9755 aaagtttaaa aactgaaccg tatgcagagt tccgaagcaa gtcaagtttg taaatgcata 9815 cctaaaaata tttaataaac gatgcagaat cct 9848 4 2749 PRT Mus musculus 4 Met Ser Asp Lys Met Ser Ser Phe Leu His Ile Gly Asp Ile Cys Ser 1 5 10 15 Leu Tyr Ala Glu Gly Ser Thr Asn Gly Phe Ile Ser Thr Leu Gly Leu 20 25 30 Val Asp Asp Arg Cys Val Val Gln Pro Glu Ala Gly Asp Leu Asn Asn 35 40 45 Pro Pro Lys Lys Phe Arg Asp Cys Leu Phe Lys Leu Cys Pro Met Asn 50 55 60 Arg Tyr Ser Ala Gln Lys Gln Phe Trp Lys Ala Ala Lys Pro Gly Ala 65 70 75 80 Asn Ser Thr Thr Asp Ala Val Leu Leu Asn Lys Leu His His Ala Ala 85 90 95 Asp Leu Glu Lys Lys Gln Asn Glu Thr Glu Asn Arg Lys Leu Leu Gly 100 105 110 Thr Val Ile Gln Tyr Gly Asn Val Ile Gln Leu Leu His Leu Lys Ser 115 120 125 Asn Lys Tyr Leu Thr Val Asn Lys Arg Leu Pro Ala Leu Leu Glu Lys 130 135 140 Asn Ala Met Arg Val Thr Leu Asp Glu Ala Gly Asn Glu Gly Ser Trp 145 150 155 160 Phe Tyr Ile Gln Pro Phe Tyr Lys Leu Arg Ser Ile Gly Asp Ser Val 165 170 175 Val Ile Gly Asp Lys Val Val Leu Asn Pro Val Asn Ala Gly Gln Pro 180 185 190 Leu His Ala Ser Ser His Gln Leu Val Asp Asn Pro Gly Cys Asn Glu 195 200 205 Val Asn Ser Val Asn Cys Asn Thr Ser Trp Lys Ile Val Leu Phe Met 210 215 220 Lys Trp Ser Asp Asn Lys Asp Asp Ile Leu Lys Gly Gly Asp Val Val 225 230 235 240 Arg Leu Phe His Ala Glu Gln Glu Lys Phe Leu Thr Cys Asp Glu His 245 250 255 Arg Lys Lys Gln His Val Phe Leu Arg Thr Thr Gly Arg Gln Ser Ala 260 265 270 Thr Ser Ala Thr Ser Ser Lys Ala Leu Trp Glu Val Glu Val Val Gln 275 280 285 His Asp Pro Cys Arg Gly Gly Ala Gly Tyr Trp Asn Ser Leu Phe Arg 290 295 300 Phe Lys His Leu Ala Thr Gly His Tyr Leu Ala Ala Glu Val Asp Pro 305 310 315 320 Asp Phe Glu Glu Glu Cys Leu Glu Phe Gln Pro Ser Val Asp Pro Asp 325 330 335 Gln Asp Ala Ser Arg Ser Arg Leu Arg Asn Ala Gln Glu Lys Met Val 340 345 350 Tyr Ser Leu Val Ser Val Pro Glu Gly Asn Asp Ile Ser Ser Ile Phe 355 360 365 Glu Leu Asp Pro Thr Thr Leu Arg Gly Gly Asp Ser Leu Val Pro Arg 370 375 380 Asn Ser Tyr Val Arg Leu Arg His Leu Cys Thr Asn Thr Trp Val His 385 390 395 400 Ser Thr Asn Ile Pro Ile Asp Lys Glu Glu Glu Lys Pro Val Met Leu 405 410 415 Lys Ile Gly Thr Ser Pro Leu Lys Glu Asp Lys Glu Ala Phe Ala Ile 420 425 430 Val Pro Val Ser Pro Ala Glu Val Arg Asp Leu Asp Phe Ala Asn Asp 435 440 445 Ala Ser Lys Val Leu Gly Ser Ile Ala Gly Lys Leu Glu Lys Gly Thr 450 455 460 Ile Thr Gln Asn Glu Arg Arg Ser Val Thr Lys Leu Leu Glu Asp Leu 465 470 475 480 Val Tyr Phe Val Thr Gly Gly Thr Asn Ser Gly Gln Asp Val Leu Glu 485 490 495 Val Val Phe Ser Lys Pro Asn Arg Glu Arg Gln Lys Leu Met Arg Glu 500 505 510 Gln Asn Ile Leu Lys Gln Ile Phe Lys Leu Leu Gln Ala Pro Phe Thr 515 520 525 Asp Cys Gly Asp Gly Pro Met Leu Arg Leu Glu Glu Leu Gly Asp Gln 530 535 540 Arg His Ala Pro Phe Arg His Ile Cys Arg Leu Cys Tyr Arg Val Leu 545 550 555 560 Arg His Ser Gln Gln Asp Tyr Arg Lys Asn Gln Glu Tyr Ile Ala Lys 565 570 575 Gln Phe Gly Phe Met Gln Lys Gln Ile Gly Tyr Asp Val Leu Ala Glu 580 585 590 Asp Thr Ile Thr Ala Leu Leu His Asn Asn Arg Lys Leu Leu Glu Lys 595 600 605 His Ile Thr Ala Ala Glu Ile Asp Thr Phe Val Ser Leu Val Arg Lys 610 615 620 Asn Arg Glu Pro Arg Phe Leu Asp Tyr Leu Ser Asp Leu Cys Val Ser 625 630 635 640 Met Asn Lys Ser Ile Pro Val Thr Gln Glu Leu Ile Cys Lys Ala Val 645 650 655 Leu Asn Pro Thr Asn Ala Asp Ile Leu Ile Glu Thr Lys Leu Val Leu 660 665 670 Ser Arg Phe Glu Phe Glu Gly Val Ser Thr Gly Glu Asn Ala Leu Glu 675 680 685 Ala Gly Glu Asp Glu Glu Glu Val Trp Leu Phe Trp Arg Asp Ser Asn 690 695 700 Lys Glu Ile Arg Ser Lys Ser Val Arg Glu Leu Ala Gln Asp Ala Lys 705 710 715 720 Glu Gly Gln Lys Glu Asp Arg Asp Ile Leu Ser Tyr Tyr Arg Tyr Gln 725 730 735 Leu Asn Leu Phe Ala Arg Met Cys Leu Asp Arg Gln Tyr Leu Ala Ile 740 745 750 Asn Glu Ile Ser Gly Gln Leu Asp Val Asp Leu Ile Leu Arg Cys Met 755 760 765 Ser Asp Glu Asn Leu Pro Tyr Asp Leu Arg Ala Ser Phe Cys Arg Leu 770 775 780 Met Leu His Met His Val Asp Arg Asp Pro Gln Glu Gln Val Thr Pro 785 790 795 800 Val Lys Tyr Ala Arg Leu Trp Ser Glu Ile Pro Ser Glu Ile Ala Ile 805 810 815 Asp Asp Tyr Asp Ser Ser Gly Thr Ser Lys Asp Glu Ile Lys Glu Arg 820 825 830 Phe Ala Gln Thr Met Glu Phe Val Glu Glu Tyr Leu Arg Asp Val Val 835 840 845 Cys Gln Arg Phe Pro Phe Ser Asp Lys Glu Lys Asn Lys Leu Thr Phe 850 855 860 Glu Val Val Asn Leu Ala Arg Asn Leu Ile Tyr Phe Gly Phe Tyr Asn 865 870 875 880 Phe Ser Asp Leu Leu Arg Leu Thr Lys Ile Leu Leu Ala Ile Leu Asp 885 890 895 Cys Val His Val Thr Thr Ile Phe Pro Ile Ser Lys Met Thr Lys Gly 900 905 910 Glu Glu Asn Lys Gly Ser Asn Val Met Arg Ser Ile His Gly Val Gly 915 920 925 Glu Leu Met Thr Gln Val Val Leu Arg Gly Gly Gly Phe Leu Pro Met 930 935 940 Thr Pro Met Ala Ala Ala Pro Glu Gly Asn Val Lys Gln Ala Glu Pro 945 950 955 960 Glu Lys Glu Asp Ile Met Val Met Asp Thr Lys Leu Lys Ile Ile Glu 965 970 975 Ile Leu Gln Phe Ile Leu Asn Val Arg Leu Asp Tyr Arg Ile Ser Cys 980 985 990 Leu Leu Cys Ile Phe Lys Arg Glu Phe Asp Glu Ser Asn Ser Gln Ser 995 1000 1005 Ser Glu Thr Ser Ser Gly Asn Ser Ser Gln Glu Gly Pro Ser Asn Val 1010 1015 1020 Pro Gly Ala Leu Asp Phe Glu His Ile Glu Glu Gln Ala Glu Gly Ile 1025 1030 1035 1040 Phe Gly Gly Ser Glu Glu Asn Thr Pro Leu Asp Leu Asp Asp His Gly 1045 1050 1055 Gly Arg Thr Phe Leu Arg Val Leu Leu His Leu Thr Met His Asp Tyr 1060 1065 1070 Pro Pro Leu Val Ser Gly Ala Leu Gln Leu Leu Phe Arg His Phe Ser 1075 1080 1085 Gln Arg Gln Glu Val Leu Gln Ala Phe Lys Gln Val Gln Leu Leu Val 1090 1095 1100 Thr Ser Gln Asp Val Asp Asn Tyr Lys Gln Ile Lys Gln Asp Leu Asp 1105 1110 1115 1120 Gln Leu Arg Ser Ile Val Glu Lys Ser Glu Leu Trp Val Tyr Lys Gly 1125 1130 1135 Gln Gly Pro Asp Glu Pro Met Asp Gly Ala Ser Gly Glu Asn Glu His 1140 1145 1150 Lys Lys Thr Glu Glu Gly Thr Ser Lys Pro Leu Lys His Glu Ser Thr 1155 1160 1165 Ser Ser Tyr Asn Tyr Arg Val Val Lys Glu Ile Leu Ile Arg Leu Ser 1170 1175 1180 Lys Leu Cys Val Gln Glu Ser Ala Ser Val Arg Lys Ser Arg Lys Gln 1185 1190 1195 1200 Gln Gln Arg Leu Leu Arg Asn Met Gly Ala His Ala Val Val Leu Glu 1205 1210 1215 Leu Leu Gln Ile Pro Tyr Glu Lys Ala Glu Asp Thr Lys Met Gln Glu 1220 1225 1230 Ile Met Arg Leu Ala His Glu Phe Leu Gln Asn Phe Cys Ala Gly Asn 1235 1240 1245 Gln Gln Asn Gln Ala Leu Leu His Lys His Ile Asn Leu Phe Leu Lys 1250 1255 1260 Pro Gly Ile Leu Glu Ala Val Thr Met Gln His Ile Phe Met Asn Asn 1265 1270 1275 1280 Phe Gln Leu Cys Ser Glu Ile Asn Glu Arg Val Val Gln His Phe Val 1285 1290 1295 His Cys Ile Glu Thr His Gly Arg Asn Val Gln Tyr Ile Lys Phe Leu 1300 1305 1310 Gln Thr Ile Val Lys Ala Glu Gly Lys Phe Ile Lys Lys Cys Gln Asp 1315 1320 1325 Met Val Met Ala Glu Leu Val Asn Ser Gly Glu Asp Val Leu Val Phe 1330 1335 1340 Tyr Asn Asp Arg Ala Ser Phe Gln Thr Leu Ile Gln Met Met Arg Ser 1345 1350 1355 1360 Glu Arg Asp Arg Met Asp Glu Asn Ser Pro Leu Met Tyr His Ile His 1365 1370 1375 Leu Val Glu Leu Leu Ala Val Cys Thr Glu Gly Lys Asn Val Tyr Thr 1380 1385 1390 Glu Ile Lys Cys Asn Ser Leu Leu Pro Leu Asp Asp Ile Val Arg Val 1395 1400 1405 Val Thr His Glu Asp Cys Ile Pro Glu Val Lys Ile Ala Tyr Ile Asn 1410 1415 1420 Phe Leu Asn His Cys Tyr Val Asp Thr Glu Val Glu Met Lys Glu Ile 1425 1430 1435 1440 Tyr Thr Ser Asn His Met Trp Lys Leu Phe Glu Asn Phe Leu Val Asp 1445 1450 1455 Ile Cys Arg Ala Cys Asn Asn Thr Ser Asp Arg Lys His Ala Asp Ser 1460 1465 1470 Ile Leu Glu Lys Tyr Val Thr Glu Ile Val Met Ser Ile Val Thr Thr 1475 1480 1485 Phe Phe Ser Ser Pro Phe Ser Asp Gln Ser Thr Thr Leu Gln Thr Arg 1490 1495 1500 Gln Pro Val Phe Val Gln Leu Leu Gln Gly Val Phe Arg Val Tyr His 1505 1510 1515 1520 Cys Asn Trp Leu Met Pro Ser Gln Lys Ala Ser Val Glu Ser Cys Ile 1525 1530 1535 Arg Val Leu Ser Asp Val Ala Lys Ser Arg Ala Ile Ala Ile Pro Val 1540 1545 1550 Asp Leu Asp Ser Gln Val Asn Asn Leu Phe Leu Lys Ser His Asn Ile 1555 1560 1565 Val Gln Lys Thr Ala Leu Asn Trp Arg Leu Ser Ala Arg Asn Ala Ala 1570 1575 1580 Arg Arg Asp Ser Val Leu Ala Ala Ser Arg Asp Tyr Arg Asn Ile Ile 1585 1590 1595 1600 Glu Arg Leu Gln Asp Ile Val Ser Ala Leu Glu Asp Arg Leu Arg Pro 1605 1610 1615 Leu Val Gln Ala Glu Leu Ser Val Leu Val Asp Val Leu His Arg Pro 1620 1625 1630 Glu Leu Leu Phe Pro Glu Asn Thr Asp Ala Arg Arg Lys Cys Glu Ser 1635 1640 1645 Gly Gly Phe Ile Cys Lys Leu Ile Lys His Thr Lys Gln Leu Leu Glu 1650 1655 1660 Glu Asn Glu Glu Lys Leu Cys Ile Lys Val Leu Gln Thr Leu Arg Glu 1665 1670 1675 1680 Met Met Thr Lys Asp Arg Gly Tyr Gly Glu Lys Gln Ile Ser Ile Asp 1685 1690 1695 Glu Ser Glu Asn Ala Glu Leu Pro Gln Ala Pro Glu Ala Glu Asn Ser 1700 1705 1710 Thr Glu Gln Glu Leu Glu Pro Ser Pro Pro Leu Arg Gln Leu Glu Asp 1715 1720 1725 His Lys Arg Gly Glu Ala Leu Arg Gln Ile Leu Val Asn Arg Tyr Tyr 1730 1735 1740 Gly Asn Ile Arg Pro Ser Gly Arg Arg Glu Ser Leu Thr Ser Phe Gly 1745 1750 1755 1760 Asn Gly Pro Leu Ser Pro Gly Gly Pro Ser Lys Pro Gly Gly Gly Gly 1765 1770 1775 Gly Gly Pro Gly Ser Ser Ser Thr Ser Arg Gly Glu Met Ser Leu Ala 1780 1785 1790 Glu Val Gln Cys His Leu Asp Lys Glu Gly Ala Ser Asn Leu Val Ile 1795 1800 1805 Asp Leu Ile Met Asn Ala Ser Ser Asp Arg Val Phe His Glu Ser Ile 1810 1815 1820 Leu Leu Ala Ile Ala Leu Leu Glu Gly Gly Asn Thr Thr Ile Gln His 1825 1830 1835 1840 Ser Phe Phe Cys Arg Leu Thr Glu Asp Lys Lys Ser Glu Lys Phe Phe 1845 1850 1855 Lys Val Phe Tyr Asp Arg Met Lys Val Ala Gln Gln Glu Ile Lys Ala 1860 1865 1870 Thr Val Thr Val Asn Thr Ser Asp Leu Gly Asn Lys Lys Lys Asp Asp 1875 1880 1885 Glu Val Asp Arg Asp Ala Pro Ser Arg Lys Lys Ala Lys Glu Pro Thr 1890 1895 1900 Thr Gln Ile Thr Glu Glu Val Arg Asp Gln Leu Leu Glu Ala Ser Ala 1905 1910 1915 1920 Ala Thr Arg Lys Ala Phe Thr Thr Phe Arg Arg Glu Ala Asp Pro Asp 1925 1930 1935 Asp His Tyr Gln Ser Gly Glu Gly Thr Gln Ala Thr Thr Asp Lys Ala 1940 1945 1950 Lys Asp Asp Leu Glu Met Ser Ala Val Ile Thr Ile Met Gln Pro Ile 1955 1960 1965 Leu Arg Phe Leu Gln Leu Leu Cys Glu Asn His Asn Arg Asp Leu Gln 1970 1975 1980 Asn Phe Leu Arg Cys Gln Asn Asn Lys Thr Asn Tyr Asn Leu Val Cys 1985 1990 1995 2000 Glu Thr Leu Gln Phe Leu Asp Cys Ile Cys Gly Ser Thr Thr Gly Gly 2005 2010 2015 Leu Gly Leu Leu Gly Leu Tyr Ile Asn Glu Lys Asn Val Ala Leu Ile 2020 2025 2030 Asn Gln Thr Leu Glu Ser Leu Thr Glu Tyr Cys Gln Gly Pro Cys His 2035 2040 2045 Glu Asn Gln Asn Cys Ile Ala Thr His Glu Ser Asn Gly Ile Asp Ile 2050 2055 2060 Ile Thr Ala Leu Ile Leu Asn Asp Ile Asn Pro Leu Gly Lys Lys Arg 2065 2070 2075 2080 Met Asp Leu Val Leu Glu Leu Lys Asn Asn Ala Ser Lys Leu Leu Leu 2085 2090 2095 Ala Ile Met Glu Ser Arg His Asp Ser Glu Asn Ala Glu Arg Ile Leu 2100 2105 2110 Tyr Asn Met Arg Pro Lys Glu Leu Val Glu Val Ile Lys Lys Ala Tyr 2115 2120 2125 Met Gln Gly Glu Val Glu Phe Glu Asp Gly Glu Asn Gly Glu Asp Gly 2130 2135 2140 Ala Ala Ser Pro Arg Asn Val Gly His Asn Ile Tyr Ile Leu Ala His 2145 2150 2155 2160 Gln Leu Ala Arg His Asn Lys Glu Leu Gln Thr Met Leu Lys Pro Gly 2165 2170 2175 Gly Gln Val Asp Gly Asp Glu Ala Leu Glu Phe Tyr Ala Lys His Thr 2180 2185 2190 Ala Gln Ile Glu Ile Val Arg Leu Asp Arg Thr Met Glu Gln Ile Val 2195 2200 2205 Phe Pro Val Pro Ser Ile Cys Glu Phe Leu Thr Lys Glu Ser Lys Leu 2210 2215 2220 Arg Ile Tyr Tyr Thr Thr Glu Arg Asp Glu Gln Gly Ser Lys Ile Asn 2225 2230 2235 2240 Asp Phe Phe Leu Arg Ser Glu Asp Leu Phe Asn Glu Met Asn Trp Gln 2245 2250 2255 Lys Lys Leu Arg Ala Gln Pro Val Leu Tyr Trp Cys Ala Arg Asn Met 2260 2265 2270 Ser Phe Trp Ser Ser Ile Ser Phe Asn Leu Ala Val Leu Met Asn Leu 2275 2280 2285 Leu Val Ala Phe Phe Tyr Pro Phe Lys Gly Val Arg Gly Gly Thr Leu 2290 2295 2300 Glu Pro His Trp Ser Gly Leu Leu Trp Thr Ala Met Leu Ile Ser Leu 2305 2310 2315 2320 Ala Ile Val Ile Ala Leu Pro Lys Pro His Gly Ile Arg Ala Leu Ile 2325 2330 2335 Ala Ser Thr Ile Leu Arg Leu Ile Phe Ser Val Gly Leu Gln Pro Thr 2340 2345 2350 Leu Phe Leu Leu Gly Ala Phe Asn Val Cys Asn Lys Ile Ile Phe Leu 2355 2360 2365 Met Ser Phe Val Gly Asn Cys Gly Thr Phe Thr Arg Gly Tyr Arg Ala 2370 2375 2380 Met Val Leu Asp Val Glu Phe Leu Tyr His Leu Leu Tyr Leu Leu Ile 2385 2390 2395 2400 Cys Ala Met Gly Leu Phe Val His Glu Phe Phe Tyr Ser Leu Leu Leu 2405 2410 2415 Phe Asp Leu Val Tyr Arg Glu Glu Thr Leu Leu Asn Val Ile Lys Ser 2420 2425 2430 Val Thr Arg Asn Gly Arg Ser Ile Ile Leu Thr Ala Val Leu Ala Leu 2435 2440 2445 Ile Leu Val Tyr Leu Phe Ser Ile Val Gly Tyr Leu Phe Phe Lys Asp 2450 2455 2460 Asp Phe Ile Leu Glu Val Asp Arg Leu Pro Asn Glu Thr Ala Val Pro 2465 2470 2475 2480 Glu Thr Gly Glu Ser Leu Ala Asn Asp Phe Leu Tyr Ser Asp Val Cys 2485 2490 2495 Arg Val Glu Thr Gly Glu Asn Cys Thr Ser Pro Ala Pro Lys Glu Glu 2500 2505 2510 Leu Leu Pro Ala Glu Glu Thr Glu Gln Asp Lys Glu His Thr Cys Glu 2515 2520 2525 Thr Leu Leu Met Cys Ile Val Thr Val Leu Ser His Gly Leu Arg Ser 2530 2535 2540 Gly Gly Gly Val Gly Asp Val Leu Arg Lys Pro Ser Lys Glu Glu Pro 2545 2550 2555 2560 Leu Phe Ala Ala Arg Val Ile Tyr Asp Leu Leu Phe Phe Phe Met Val 2565 2570 2575 Ile Ile Ile Val Leu Asn Leu Ile Phe Gly Val Ile Ile Asp Thr Phe 2580 2585 2590 Ala Asp Leu Arg Ser Glu Lys Gln Lys Lys Glu Glu Ile Leu Lys Thr 2595 2600 2605 Thr Cys Phe Ile Cys Gly Leu Glu Arg Asp Lys Phe Asp Asn Lys Thr 2610 2615 2620 Val Thr Phe Glu Glu His Ile Lys Glu Glu His Asn Met Trp His Tyr 2625 2630 2635 2640 Leu Cys Phe Ile Val Leu Val Lys Val Lys Asp Ser Thr Glu Tyr Thr 2645 2650 2655 Gly Pro Glu Ser Tyr Val Ala Glu Met Ile Arg Glu Arg Asn Leu Asp 2660 2665 2670 Trp Phe Leu Arg Met Arg Ala Met Ser Leu Val Ser Ser Asp Ser Glu 2675 2680 2685 Gly Glu Gln Asn Glu Leu Arg Asn Leu Gln Glu Lys Leu Glu Ser Thr 2690 2695 2700 Met Lys Leu Val Thr Asn Leu Ser Gly Gln Leu Ser Glu Leu Lys Asp 2705 2710 2715 2720 Gln Met Thr Glu Gln Arg Lys Gln Lys Gln Arg Ile Gly Leu Leu Gly 2725 2730 2735 His Pro Pro His Met Asn Val Asn Pro Gln Gln Pro Ala 2740 2745 5 23 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 5 tgtcagacat atgcgtgttg gaa 23 6 36 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 6 cgcgggatcc ttatttccgg ttgttgtgga gcaggg 36 7 35 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 7 ccggaattct tatttccggt tgttgtggag caggg 35 8 37 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 8 cgcggatcca tgaaatggag tgataacaaa gacgaca 37 9 24 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 9 gagagcggca ggcactgatg aggg 24 10 24 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 10 ccctcatcag tgcctgccgc tctc 24 11 25 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 11 gctgaggttc aagacctgga ctttg 25 12 24 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 12 aaagtccagg tcttgaacct cagc 24 13 1059 DNA Mus musculus CDS (1)..(1059) 13 tgg agt gat aac aaa gac gac att ctc aaa gga ggt gat gtg gtg agg 48 Trp Ser Asp Asn Lys Asp Asp Ile Leu Lys Gly Gly Asp Val Val Arg 1 5 10 15 ctc ttc cat gcc gag caa gag aag ttt ctc acc tgt gat gag cac cgg 96 Leu Phe His Ala Glu Gln Glu Lys Phe Leu Thr Cys Asp Glu His Arg 20 25 30 aag aag cag cat gtg ttc ctg agg acc acc ggc agg cag tca gcc acg 144 Lys Lys Gln His Val Phe Leu Arg Thr Thr Gly Arg Gln Ser Ala Thr 35 40 45 tcg gcc acc agt tct aaa gcc ctg tgg gaa gtg gag gta gtc cag cac 192 Ser Ala Thr Ser Ser Lys Ala Leu Trp Glu Val Glu Val Val Gln His 50 55 60 gac cca tgt cgg ggt gga gct ggg tac tgg aat agc ctc ttc cgg ttc 240 Asp Pro Cys Arg Gly Gly Ala Gly Tyr Trp Asn Ser Leu Phe Arg Phe 65 70 75 80 aag cac ctg gct aca ggg cat tac ttg gct gca gag gta gac cct gac 288 Lys His Leu Ala Thr Gly His Tyr Leu Ala Ala Glu Val Asp Pro Asp 85 90 95 ttt gag gaa gaa tgc ctg gag ttt cag ccc tca gtg gac cct gat cag 336 Phe Glu Glu Glu Cys Leu Glu Phe Gln Pro Ser Val Asp Pro Asp Gln 100 105 110 gat gca tct cgg agt agg ttg aga aac gcg caa gaa aaa atg gta tac 384 Asp Ala Ser Arg Ser Arg Leu Arg Asn Ala Gln Glu Lys Met Val Tyr 115 120 125 tct ctg gtc tcc gtg cct gaa ggc aac gac atc tcc tcc atc ttt gag 432 Ser Leu Val Ser Val Pro Glu Gly Asn Asp Ile Ser Ser Ile Phe Glu 130 135 140 cta gac ccc acg act ctg cgt gga ggt gac agc ctt gtc cca agg aac 480 Leu Asp Pro Thr Thr Leu Arg Gly Gly Asp Ser Leu Val Pro Arg Asn 145 150 155 160 tcc tat gtc cgt ctc aga cac ctg tgc acc aac acc tgg gta cac agc 528 Ser Tyr Val Arg Leu Arg His Leu Cys Thr Asn Thr Trp Val His Ser 165 170 175 aca aac atc ccc atc gac aag gaa gag gag aag cct gtg atg ctg aaa 576 Thr Asn Ile Pro Ile Asp Lys Glu Glu Glu Lys Pro Val Met Leu Lys 180 185 190 att ggt acc tct ccc ctg aag gag gac aag gaa gca ttt gcc ata gtt 624 Ile Gly Thr Ser Pro Leu Lys Glu Asp Lys Glu Ala Phe Ala Ile Val 195 200 205 cct gtt tcc cct gct gag gtt cgg gac ctg gac ttt gcc aat gat gcc 672 Pro Val Ser Pro Ala Glu Val Arg Asp Leu Asp Phe Ala Asn Asp Ala 210 215 220 agc aag gtg ctg ggc tcc atc gct ggg aag ttg gaa aag ggc acc atc 720 Ser Lys Val Leu Gly Ser Ile Ala Gly Lys Leu Glu Lys Gly Thr Ile 225 230 235 240 acc cag aat gag aga agg tct gtc acg aag ctt ttg gaa gac ttg gtt 768 Thr Gln Asn Glu Arg Arg Ser Val Thr Lys Leu Leu Glu Asp Leu Val 245 250 255 tac ttt gtc acg ggt gga act aac tct ggc caa gac gtg ctt gaa gtt 816 Tyr Phe Val Thr Gly Gly Thr Asn Ser Gly Gln Asp Val Leu Glu Val 260 265 270 gtc ttc tct aag ccc aat cga gag cgg cag aag ctg atg agg gaa cag 864 Val Phe Ser Lys Pro Asn Arg Glu Arg Gln Lys Leu Met Arg Glu Gln 275 280 285 aat att ctc aag cag atc ttc aag ctg ttg cag gcc ccc ttc acg gac 912 Asn Ile Leu Lys Gln Ile Phe Lys Leu Leu Gln Ala Pro Phe Thr Asp 290 295 300 tgc ggg gat ggc ccg atg ctt cgg ctg gag gag ctg ggg gat cag cgc 960 Cys Gly Asp Gly Pro Met Leu Arg Leu Glu Glu Leu Gly Asp Gln Arg 305 310 315 320 cat gct cct ttc aga cat att tgc cga ctc tgc tac agg gtc ctg cga 1008 His Ala Pro Phe Arg His Ile Cys Arg Leu Cys Tyr Arg Val Leu Arg 325 330 335 cac tca cag caa gac tac agg aag aac cag gag tac ata gcc aag cag 1056 His Ser Gln Gln Asp Tyr Arg Lys Asn Gln Glu Tyr Ile Ala Lys Gln 340 345 350 ttt 1059 Phe 14 353 PRT Mus musculus 14 Trp Ser Asp Asn Lys Asp Asp Ile Leu Lys Gly Gly Asp Val Val Arg 1 5 10 15 Leu Phe His Ala Glu Gln Glu Lys Phe Leu Thr Cys Asp Glu His Arg 20 25 30 Lys Lys Gln His Val Phe Leu Arg Thr Thr Gly Arg Gln Ser Ala Thr 35 40 45 Ser Ala Thr Ser Ser Lys Ala Leu Trp Glu Val Glu Val Val Gln His 50 55 60 Asp Pro Cys Arg Gly Gly Ala Gly Tyr Trp Asn Ser Leu Phe Arg Phe 65 70 75 80 Lys His Leu Ala Thr Gly His Tyr Leu Ala Ala Glu Val Asp Pro Asp 85 90 95 Phe Glu Glu Glu Cys Leu Glu Phe Gln Pro Ser Val Asp Pro Asp Gln 100 105 110 Asp Ala Ser Arg Ser Arg Leu Arg Asn Ala Gln Glu Lys Met Val Tyr 115 120 125 Ser Leu Val Ser Val Pro Glu Gly Asn Asp Ile Ser Ser Ile Phe Glu 130 135 140 Leu Asp Pro Thr Thr Leu Arg Gly Gly Asp Ser Leu Val Pro Arg Asn 145 150 155 160 Ser Tyr Val Arg Leu Arg His Leu Cys Thr Asn Thr Trp Val His Ser 165 170 175 Thr Asn Ile Pro Ile Asp Lys Glu Glu Glu Lys Pro Val Met Leu Lys 180 185 190 Ile Gly Thr Ser Pro Leu Lys Glu Asp Lys Glu Ala Phe Ala Ile Val 195 200 205 Pro Val Ser Pro Ala Glu Val Arg Asp Leu Asp Phe Ala Asn Asp Ala 210 215 220 Ser Lys Val Leu Gly Ser Ile Ala Gly Lys Leu Glu Lys Gly Thr Ile 225 230 235 240 Thr Gln Asn Glu Arg Arg Ser Val Thr Lys Leu Leu Glu Asp Leu Val 245 250 255 Tyr Phe Val Thr Gly Gly Thr Asn Ser Gly Gln Asp Val Leu Glu Val 260 265 270 Val Phe Ser Lys Pro Asn Arg Glu Arg Gln Lys Leu Met Arg Glu Gln 275 280 285 Asn Ile Leu Lys Gln Ile Phe Lys Leu Leu Gln Ala Pro Phe Thr Asp 290 295 300 Cys Gly Asp Gly Pro Met Leu Arg Leu Glu Glu Leu Gly Asp Gln Arg 305 310 315 320 His Ala Pro Phe Arg His Ile Cys Arg Leu Cys Tyr Arg Val Leu Arg 325 330 335 His Ser Gln Gln Asp Tyr Arg Lys Asn Gln Glu Tyr Ile Ala Lys Gln 340 345 350 Phe 

What is claimed is:
 1. A recombinant polypeptide of the following (a), (b) or (c): (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 2; (b) a polypeptide comprising an amino acid sequence having deletion, substitution or addition of at least one amino acid in the amino acid sequence shown in SEQ ID NO: 2, and having a high affinity binding activity to inositol 1,4,5-trisphosphate; or (c) a polypeptide having at least 70% homology with the amino acid sequence shown in SEQ ID NO: 2, and having a high affinity binding activity with inositol 1,4,5-trisphosphate.
 2. An antagonist for IP₃-induced calcium comprising the protein of claim
 1. 3. A therapeutic agent diseases associated with calcium production comprising the protein of claim
 1. 4. An agent for gene therapy for diseases associated with calcium production comprising the gene of claims 2 or
 3. 5. The therapeutic agent of claim 4, wherein the disease is at least one disease selected from the group consisting of diseases in the nervous system, blood vascular system, respiratory system, digestive system, lymphatic system, urinary system and reproduction system. 